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The Repair and Tolerance of DNA Damage in
Higher Plants
by
Edward J. Vonarx, B.Sc
Submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
Deakin University
August, 2000
ii
DEAKIN UNIVERSITY
CANDIDATE DECLARATION
I certify that the thesis entitled:
The Repair and Tolerance of DNA Damage in Higher Plants
submitted for the degree of:
Doctor of Philosophy
is the result of my own research, except where otherwise acknowledged, and that this
thesis in whole or in part has not been submitted for an award, including a higher
degree, to any other university or institution.
Full Name: Edward Joseph Vonarx
Signed: ..............................................................................…………………
Date: .
iii
This thesis may be made available for consultation, loan and limited copying in
accordance with the Copyright Act 1968.
iv
Abstract
DNA repair mechanisms constitute an essential cellular response to DNA
damage arising either from metabolic processes or from environmental sources such
as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by
processes such as nucleotide excision repair (NER), a coordinated pathway in which
lesions and the surrounding nucleotides are excised and replaced via DNA
resynthesis. The importance of repair is illustrated by human disease states such as
xeroderma pigmentosum and Cockayne’s syndrome which result from defects in the
NER system arising from mutations in XP- genes or XP- and CS- genes respectively
Little detail is known of DNA damage repair processes in plants, despite the
economic and ecological importance of these organisms. This study aimed to expand
our knowledge of the process of NER in plants, largely via a polymerase chain
reaction (PCR)-based approach involving amplification, cloning and characterisation
of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1
and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from
the model dicotyledonous plant Arabidopsis thaliana. The sequence of the
3’-untranslated region of atXPD was also determined. Comparison of genomic and
cDNA sequences allowed a detailed analysis of gene structures, including details of
intron/exon processing. Variable transcript processing to produce three distinct
transcripts was found in the case of atXPF. In an attempt to validate the proposed
homologous function of these cDNAs, assays to test complementation of resistance
to ultraviolet radiation in the relevant yeast mutants were performed. Despite
extensive amino acid sequence conservation, neither plant cDNA was able to restore
UV-resistance. As the yeast RAD3 gene product is also involved in vivo in
transcription, and so is required for viability, the atXPD cDNA was tested in a
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complementation assay for this function in an appropriate yeast mutant. The plant
cDNA was found to substantially increase the viability of the yeast mutant.
The structural and functional significance of these results is discussed
comparatively with reference to yeast, human and other known homologues. Other
putative NER homologues were identified in A. thaliana database sequences,
including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects
of ongoing investigations. This study also describes preliminary investigations of
putative REV3 and RAD30 translesion synthesis genes from A. thaliana.
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Table of Contents
ABSTRACT ..........................................................................................................................IV
LIST OF FIGURES .............................................................................................................. X
LIST OF TABLES ............................................................................................................. XII
ACKNOWLEDGEMENTS..............................................................................................XIII
ABBREVIATIONS ...........................................................................................................XIV
1 INTRODUCTION............................................................................................................... 1
1.1 THE REQUIREMENT FOR DNA REPAIR IN HIGHER PLANTS............................................... 2
1.1.1 Seeds and pollen .................................................................................................... 2
1.1.2 UV-radiation and the induction of DNA damage .................................................. 4
1.1.3 UV-radiation effects on plants ............................................................................... 5
1.2 INFLUENCE OF PLANT DEVELOPMENT ON DNA REPAIR MECHANISMS ............................ 6
1.3 PRIMARY RESPONSES TO EXTERNAL SOURCES OF DNA DAMAGE ................................... 8
1.4 SECONDARY RESPONSES TO SPONTANEOUS AND INDUCED DNA DAMAGE ..................... 8
1.4.1 Photoreactivation................................................................................................... 9
1.4.2 Base excision repair (BER).................................................................................. 11
1.4.3 Nucleotide excision repair (NER)........................................................................ 13
1.4.3.1 Human and yeast nucleotide excision repair ...............................................................13
1.4.3.2 NER proteins and cell cycle progression in humans ...................................................20
1.4.3.3 NER proteins and transcription-coupled repair ...........................................................21
1.4.3.4 Plant nucleotide excision repair ..................................................................................22
1.4.4 Mismatch correction ............................................................................................ 25
1.4.5 DNA damage tolerance........................................................................................ 27
1.4.5.1 Translesion synthesis in yeast and humans .................................................................29
1.4.5.2 DNA damage tolerance in plants ................................................................................32
1.4.6 Recombinational repair ....................................................................................... 33
1.5 RADIATION-SENSITIVE ARABIDOPSIS MUTANTS............................................................. 35
1.6 A. THALIANA AS AN EXPERIMENTAL ORGANISM ............................................................. 39
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1.7 CONCLUDING COMMENTS ............................................................................................. 40
1.8 FOCUS AND SCOPE OF THIS STUDY ................................................................................ 41
2 MATERIALS AND METHODS...................................................................................... 45
2.1 ARABIDOPSIS THALIANA ECOTYPES AND SEED LINES ...................................................... 45
2.2 ARABIDOPSIS THALIANA GROWTH CONDITIONS............................................................... 45
2.3 MEDIA .......................................................................................................................... 45
2.3.1 At -medium, derived from .................................................................................... 45
2.3.2 Luria-Bertani (LB) broth ..................................................................................... 46
2.3.3 YT broth ............................................................................................................... 46
2.3.4 2xYT broth ........................................................................................................... 46
2.3.5 YPD broth ............................................................................................................ 47
2.3.6 SD (synthetic dextrose) ........................................................................................ 47
2.3.7 Raffinose expression medium............................................................................... 47
2.3.8 SOC broth ............................................................................................................ 47
2.3.9 SD-Leu-Trp+FOA selection plates ...................................................................... 48
2.4 YEAST AND BACTERIAL STRAINS ................................................................................. 48
2.5 PLASMIDS ..................................................................................................................... 49
2.6 ISOLATION OF GENOMIC DNA FROM ARABIDOPSIS THALIANA........................................ 49
2.6.1 Method 1 .............................................................................................................. 50
2.6.2 Method 2 .............................................................................................................. 50
2.7 DNA RESTRICTION DIGESTS ......................................................................................... 51
2.8 INTERNET-BASED UTILITIES.......................................................................................... 51
2.9 SEQUENCE COMPARISONS............................................................................................. 52
2.10 POLYMERASE CHAIN REACTION PRIMERS ................................................................... 52
2.11 AMPLIFICATION BY PCR ............................................................................................ 52
2.12 PRECIPITATION OF PCR PRODUCTS............................................................................. 53
2.13 ISOLATION OF RNA FROM ARABIDOPSIS THALIANA AND SYNTHESIS OF CDNA............ 53
2.14 PREPARATION OF RNA AND CDNA FROM YEAST (SACCHAROMYCES CEREVISIAE) ...... 54
2.15 DNA SEQUENCING ..................................................................................................... 54
2.16 ATXPD 3’ RACE ....................................................................................................... 55
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2.17 YEAST TRANSFORMATION.......................................................................................... 55
2.18 UV-SENSITIVITY COMPLEMENTATION STUDIES IN YEAST........................................... 56
3 RESULTS........................................................................................................................... 59
3.1 CDNA LIBRARY SCREENING FOR AN A. THALIANA XPD HOMOLOGUE ........................... 59
3.2 VIRTUAL SCREENING FOR AN A. THALIANA XPD HOMOLOGUE ...................................... 60
3.3 CHARACTERISATION OF THE ATXPD GENOMIC, CDNA AND AMINO ACID SEQUENCES.. 61
3.3.1 atXPD 3’-RACE amplification and sequencing................................................... 62
3.4 TRANSLATION INITIATION IN ATXPD ............................................................................ 63
3.5 ATXPD COMPLEMENTATION ASSAY FOR UV-SENSITIVITY IN YEAST............................. 64
3.6 ATXPD COMPLEMENTATION ASSAY FOR VIABILITY IN YEAST ....................................... 65
3.7 VIRTUAL SCREENING FOR AN A. THALIANA XPF HOMOLOGUE....................................... 71
3.8 PCR AMPLIFICATION AND CLONING OF AN A. THALIANA XPF HOMOLOGUE .................. 71
3.9 ATXPF EXHIBITS VARIABLE TRANSCRIPT PROCESSING.................................................. 73
3.9.1 Structural features of atXPF cDNAs.................................................................... 74
3.9.2 Translation initiation in atXPF............................................................................ 75
3.10 CONFIRMATION OF THE NUCLEOTIDE SEQUENCE OF YEAST RAD1 .............................. 76
3.11 ATXPF COMPLEMENTATION ASSAYS FOR UV-SENSITIVITY IN YEAST ......................... 77
3.11.1 atXPF UV-sensitivity assay results .................................................................... 78
3.12 ASSESSMENT OF CODON USEAGE BY ATXPD AND ATXPF ........................................... 79
3.13 ISOLATION OF A RAD10/ERCC1 HOMOLOGUE FROM ARABIDOPSIS THALIANA.............. 80
3.14 ISOLATION OF AN A. THALIANA PARTIAL CDNA HOMOLOGOUS TO RAD2/XPG ......... 81
3.14.1 PCR amplification and cloning of a partial atXPG cDNA ................................ 82
3.15 TRANSLESION SYNTHESIS GENES FROM A. THALIANA .................................................. 83
3.15.1 Isolation of a RAD30/XPV homologue from Arabidopsis thaliana .................... 83
3.15.2 Isolation of a REV3 homologue from Arabidopsis thaliana ............................... 84
4 DISCUSSION .................................................................................................................... 87
4.1 EVIDENCE FOR A HOMOLOGOUS ROLE FOR ATXPD ....................................................... 87
4.1.1 RAD3/XPD mutations affecting repair ................................................................ 87
4.1.2 RAD3/XPD mutations affecting viability ............................................................. 91
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4.2 ATXPD IS AN ARABIDOPSIS RAD3/XPD HOMOLOGUE ................................................... 95
4.3 ATXPF IS AN ARABIDOPSIS RAD1/XPF HOMOLOGUE .................................................... 99
4.4 EVIDENCE FOR A HOMOLOGOUS ROLE FOR ATERCC1................................................. 103
4.5 EVIDENCE FOR A HOMOLOGOUS ROLE FOR ATXPG ..................................................... 105
4.6 COORDINATION OF NER IN PLANTS............................................................................ 107
4.7 NER AND CELL CYCLE CONTROL LINKAGES IN PLANTS .............................................. 108
4.8 NER SYSTEMS IN HIGHER PLANTS .............................................................................. 110
4.9 TLS SYSTEMS IN HIGHER PLANTS ............................................................................... 112
5 FURTHER WORK ......................................................................................................... 114
6 RECENT PUBLICATIONS........................................................................................... 116
7 BIBLIOGRAPHY ........................................................................................................... 118
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List of Figures
FIGURE 1.1 SCHEMATIC DEPICTION OF YEAST NER PROTEINS AT A DNA LESION. ....................................
....................................................................................................................... FOLLOWING PAGE 15
FIGURE 2.1 ATXPD 3’ RACE STRATEGY.................................................................. FOLLOWING PAGE 55
FIGURE 3.1 MULTIPLE ALIGNMENT OF RAD3/XPD PROTEIN HOMOLOGUES. ........... FOLLOWING PAGE 59
FIGURE 3.2 ATXPD GENOMIC, CDNA AND PROTEIN SEQUENCES. ............................ FOLLOWING PAGE 61
FIGURE 3.3 ATXPD PROTEIN ALIGNMENT. ............................................................... FOLLOWING PAGE 62
FIGURE 3.4 UV-SENSITIVITY COMPLEMENTATION OF THE RAD3-1 MUTATION.......... FOLLOWING PAGE 65
FIGURE 3.5 ATXPD VIABILITY COMPLEMENTATION ASSAY IN RAD3TS14. .................. FOLLOWING PAGE 67
FIGURE 3.6 GROWTH CURVES OF THE RAD3TS14, RAD3TS14 RAD3, AND RAD3TS14 ATXPD STRAINS AT
39OC. .............................................................................................................. FOLLOWING PAGE 69
FIGURE 3.7 GROWTH CURVES OF THE RAD3TS14, RAD3TS14 RAD3, AND RAD3TS14 ATXPD STRAINS AT
30OC. .............................................................................................................. FOLLOWING PAGE 69
FIGURE 3.8 MULTIPLE PROTEIN ALIGNMENT OF RAD1 HOMOLOGUES ..................... FOLLOWING PAGE 71
FIGURE 3.9 MULTIPLE ALIGNMENT OF SELECTED REGIONS OF UVH1 AND ATXPF CDNA SEQUENCES
....................................................................................................................... FOLLOWING PAGE 73
FIGURE 3.10 ATXPF GENE STRUCTURE .................................................................... FOLLOWING PAGE 73
FIGURE 3.11 ATXPF-3 CDNA AND PROTEIN SEQUENCE ........................................... FOLLOWING PAGE 73
FIGURE 3.12 MULTIPLE SEQUENCE ALIGNMENT OF PREDICTED ATXPF AND UVH1 PROTEIN SEQUENCES.
....................................................................................................................... FOLLOWING PAGE 74
FIGURE 3.13 MULTIPLE PROTEIN ALIGNMENT OF RAD1 AND RAD1 HOMOLOGUES INCLUDING
ATXPF-3......................................................................................................... FOLLOWING PAGE 74
FIGURE 3.14 UV-C SENSITIVITY OF YEAST WILD-TYPE AND RAD1 MUTANTS............ FOLLOWING PAGE 78
FIGURE 3.15 MULTIPLE PROTEIN ALIGNMENT OF RAD10 HOMOLOGUES. ................ FOLLOWING PAGE 81
FIGURE 3.16 ATXPG PARTIAL CDNA AND PROTEIN SEQUENCES. ............................. FOLLOWING PAGE 83
FIGURE 3.17 MULTIPLE ALIGNMENT OF PARTIAL RAD2/XPG HOMOLOGUES. ......... FOLLOWING PAGE 83
FIGURE 3.18 MULTIPLE PROTEIN ALIGNMENT OF RAD30 HOMOLOGUES. ................ FOLLOWING PAGE 84
FIGURE 3.19 MULTIPLE PROTEIN ALIGNMENT OF REV3 HOMOLOGUES.................... FOLLOWING PAGE 85
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FIGURE 4.1. POSSIBLE PHYLOGENETIC RELATIONSHIPS BETWEEN RAD3/XPD HOMOLOGUES.
....................................................................................................................... FOLLOWING PAGE 95
FIGURE 4.2. POSSIBLE PHYLOGENETIC RELATIONSHIPS OF RAD1/XPF PROTEIN HOMOLOGUES.
..................................................................................................................... FOLLOWING PAGE 103
FIGURE 4.3 MULTIPLE PROTEIN ALIGNMENT OF N, I AND C REGIONS OF RAD2/XPG HOMOLOGUES.
.....................................................................................................................FOLLOWING PAGE 106
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List of Tables
TABLE 1.1 HOMOLOGOUS REPAIR AND/OR TRANSCRIPTION PROTEINS. .................... FOLLOWING PAGE 14
TABLE 1.2. POTENTIAL DNA REPAIR DEFECTIVE MUTANTS OF ARABIDOPSIS THALIANA.............................
....................................................................................................................... FOLLOWING PAGE 35
TABLE 1.3 CHARACTERISED PLANT DNA REPAIR GENES OR CDNAS. ...................... FOLLOWING PAGE 40
TABLE 2.1. PCR AND SEQUENCING PRIMERS USED IN THIS INVESTIGATION.............. FOLLOWING PAGE 52
TABLE 3.1 ATXPD EXON/INTRON SIZES .................................................................... FOLLOWING PAGE 61
TABLE 3.2. ATXPF-3 EXON/INTRON SIZES. ............................................................... FOLLOWING PAGE 74
TABLE 3.3 VARIABLE INTRON/EXON BOUNDARIES AND THEIR UTILIZATION IN ATXPF CLONES.
....................................................................................................................... FOLLOWING PAGE 75
TABLE 3.4 RESOLUTION OF DISPUTED BASES IN RAD1. ............................................ FOLLOWING PAGE 77
TABLE 3.5 CODON USAGE IN ATXPD. ....................................................................... FOLLOWING PAGE 79
TABLE 3.6 CODON USAGE IN ATXPF......................................................................... FOLLOWING PAGE 79
TABLE 4.1 SEQUENCED OR PUTATIVE PLANT NUCLEOTIDE EXCISION REPAIR GENES................................
..................................................................................................................... FOLLOWING PAGE 111
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Acknowledgements
Some of the material contained within this thesis has already appeared in
press, as listed in section 7 below. Only material of which I was primary author has
been included herein, with the exception of Table 1.2, which was compiled by Ms.
H. Mitchell, Dr. R. Karthikeyan, Dr. I. Chaterjee and myself, and is included here for
completeness.
This research program has been possible only with the assistance of a number
of organisations and individuals, for which I am grateful. These include:
Deakin University for the provision of a post-graduate scholarship;
The Australian Research Council, for funding to Professor Kunz to allow
research projects such as these;
Professor Bernard Kunz, for the opportunity to participate in this program,
financial support, and scientific guidance along the way;
Dr. Christoph Gehring for his enthusiastic encouragement as Associate
Supervisor; and
The many others travelling a similar path, for the shared experience.
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Abbreviations
(6-4) photoproduct pyrimidine(6-4)pyrimidone
Amino acid IUPAC single letter code
A alanine
C cysteine
D aspartic acid
E glutamic acid
F phenylalanine
G glycine
H histidine
I isoleucine
K lysine
L leucine
M methionine
N asparagine
P proline
Q glutamine
R arginine
S serine
T threonine
V valine
W tryptophan
Y tyrosine
X any amino acid
bp base-pair(s)
BER base excision repair
CDS coding DNA sequence
CPD cyclobutane pyrimidine dimer
CS Cockayne’s syndrome
DNA deoxyribonucleic acid
ds double-stranded
EB extraction buffer
xv
g gram
h hour(s)
IPTG isopropyl-β-D-thiogalactoside
J/m2 joules/metre2
Kbp kilobase-pairs
m metre
M molar
Mbp megabase-pairs
MMR mismatch repair
Medium supplements
Ade adenine
Ura uracil
Tryp tryptophan
His histidine
Leu leucine
Lys lysine
min minute(s)
ηm nanometre (10-9 m)
N any deoxyribonucleotide
NER nucleotide excision repair
PCR polymerase chain reaction
PEG polyethylene glycol
RACE rapid amplification of cDNA ends
RNA ribonucleic acid
RNAse H ribonuclease H
RT room temperature (about 22oC)
s second(s)
Taq Taq DNA polymerase
TCR transcription-coupled repair
TE TE buffer
TLS translesion synthesis
TTD trichothiodystrophy
UV ultraviolet
UV-A ultraviolet radiation (320-400 nm)
xvi
UV-B ultraviolet radiation (280-320 nm)
UV-C ultraviolet radiation (100-280 nm)
xg times force of gravity
X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside
XP xeroderma pigmentosum
1
1 Introduction
Nuclear DNA is an inherently unstable molecule, and suffers spontaneous
and metabolically induced damage. Such intrinsic damage includes the loss of a base
to form an abasic site, chemical modification of a base to form a mis-coding or
non-coding lesion, and sugar-phosphate backbone breakage. DNA damage is also
generated by environmental exposure of the organism to ultraviolet (UV) and
ionising radiation, heat, desiccation and chemicals. As obligate phototrophs, and
despite shielding strategies, terrestrial plants are particularly subject to solar
UV-stress and the attendant UV-induced damage. Direct DNA damage from
excessive UV exposure includes cyclobutane pyrimidine dimers (CPDs);
pyrimidine(6-4)pyrimidone dimers ((6-4) photoproducts); DNA-protein crosslinks,
and other more rare DNA photoproducts. These lesions may result in inhibition of
DNA replication and transcription, or in a heritable mutation if not repaired before
replication (Stapleton, 1992).
Radiation-induced damage in plants can also affect physiological or
biochemical processes (Jansen et al., 1998) by such mechanisms as reduced protein
synthesis, hormone photooxidation, plasma membrane ATPase destruction and
damage to photosystem II and the thylakoid membrane (Strid et al., 1990; Stapleton,
1992). Chemical agents may cause alkylation of bases, intra- and interstrand
crosslinks, and substitution at reactive carbon or nitrogen atoms within the base
structures. Base mismatches may arise as replication errors, during recombination
events, or via the deamination of 5-methylcytosine, and also must be repaired.
Mechanisms to repair these lesions are required to maintain the necessary
genetic integrity both within an individual and, since plants continuously derive their
gametes on demand, also through each ancestral line. Failing this, the damage must
2
be tolerated in some fashion. The inability of most plants to behaviourally minimise
environmental exposure to noxious agents further increases the importance of DNA
damage avoidance and repair mechanisms. However, studies of in vivo cellular DNA
repair mechanisms have focused largely on bacteria, yeast and humans, with minor
work on other vertebrates (usually mammals) and less often other fungi, insects and
plants. Thus, it is not surprising that relatively little is known about DNA damage
repair systems in plants, or mechanisms of tolerance of DNA damage in this phylum.
The remainder of this introduction reviews DNA damage repair and tolerance
in higher plants, with reference to comparative processes in vertebrates and
particularly the model yeast, Saccharomyces cerevisiae. Although reference is made
to bacterial processes where appropriate, this review will largely be restricted to
eukaryotes and eukaryotic systems.
1.1 The requirement for DNA repair in higher plants
1.1.1 Seeds and pollen
Aside from adult plants, both pollen and seeds are subject to DNA lesions,
not only during the dehydration associated with formation and dormancy but also
from genotoxic environmental exposures. For many plant species, seed viability
decreases with seed age (subject to storage conditions), and early in the 1900s it was
observed that those older seeds retaining viability gave rise to proportionally more
mutant phenotypes than did fresh seeds. Progressive fragmentation of embryonic
nuclear DNA occurred in dry-stored seeds of barley (Elder et al., 1987).
Furthermore, a high frequency of aberrant chromosomes in the resultant plants also
corresponded with seed age (Villiers and Edgcumbe, 1975; Osborne, 1980a; Roberts,
1988; Mayer and Poljakoff-Mayber, 1989; Dimitrov, 1994). A direct correlation
3
between decreased seed viability and loss of DNA integrity has been established
from studies of chromosomal aberrations and point mutations (Murata et al., 1982;
Roberts, 1988). In addition, a type of DNA damage restricted to irradiated,
dehydrated DNA has been described. “Spore product” (5-thyminyl-6,6-
dihydrothymine) is the major lesion produced in bacterial spores under dehydrated
conditions (Jackson, 1987; McLennan, 1988). Given the dehydration inherent in the
formation of pollen and seeds, this type of DNA lesion may consequently be relevant
to studies of the maintenance of DNA integrity in plants.
Early studies of rye (Secale cereale) embryos also suggested that plants have
the ability to recover from chromosomal abnormalities over subsequent cell cycles,
and that DNA repair is one of the very early events in germination (Osborne et al.,
1980b). In a mature seed, stored dry, most embryonic cells are arrested at the G1
phase of the cell cycle (Osborne et al., 1984; Dandoy et al., 1987; Dimitrov, 1994) so
that DNA replication is an expected event before mitosis. However, unscheduled
DNA synthesis has been noted immediately after rehydration of dry seeds but before
the normal S phase DNA replication. Similarly, unscheduled DNA synthesis
associated with repair in pollen from various angiosperms has been reported
(Jackson, 1987).
DNA damage, including fragmentation of the genome, accumulated during
the seed dry-storage period presumably is repaired to some extent during this pre-S
phase period. Evidence to support this presumption includes DNA ligase activity
(Elder et al., 1987) and DNA synthesis in germinating seeds as revealed by the
incorporation of tritiated thymidine (Soyfer, 1979; Osborne, 1980a; Osborne et al.,
1984; McLennan, 1988). Furthermore, DNA replication and cell division is delayed
in seeds of low vigour/viability (Osborne, 1980a; Osborne et al., 1980b; Osborne et
4
al., 1984). For example, in a group of rye seeds of 96% viability the reported mean
delay before S phase was 4 hours, whereas in a group of seeds of 46% viability the
delay time was 11 hours, implying a delay mechanism prompted by DNA or
physiological damage. Consistent with these viability studies, younger seeds recover
faster from DNA damage than older seeds, and similarly, soaked seeds can repair
more quickly than dried seeds (Soyfer et al., 1977).
Following germination, embryonic DNA must be replicated many times to
allow the numerous cell divisions required for the seedling’s growth. This presents a
further opportunity for loss of DNA fidelity and consequent reduction in
reproductive efficiency. Empirically, variation in the viability of calluses derived
from immature embryos of barley (Hordeum vulgare) was correlated with unrepaired
chromosomal aberrations induced during tissue culture (Singh, 1986).
1.1.2 UV-radiation and the induction of DNA damage
A major focus in the study of plant DNA repair is on damage induced by
solar ultraviolet (UV) radiation. This has largely been prompted by the predicted
increase in incident UV levels associated with the deterioration of the stratospheric
ozone layer (for example, see McKenzie et al., 1999).
Ultraviolet radiation includes the wavelengths from 100 to 400 ηm, broken
into the three classes of UV-A (320-400 ηm), UV-B (280-320 ηm), and UV-C
(100-280 ηm). Solar UV at the earth’s surface consists largely of UV-A and some
UV-B, as the atmosphere is increasingly opaque to radiation of shorter wavelengths
(Friedberg et al., 1995). Experimentally, UV-C is often used for UV challenge (Kunz
et al., 1987; Friedberg et al., 1995) due to the ready availability of monochromatic
(254 ηm) sources, and the DNA absorption peak at 260 ηm. Also, the consensus
5
opinion is that the lesions formed by exposure to UV-A and UV-B are similar to
those induced in greater numbers by UV-C exposure (Friedberg et al., 1995).
CPDs and (6-4) photoproducts are the major lesions formed by exposure of
DNA to UV radiation. The frequency of formation of both types of lesion is
approximately proportional to the incident UV dose within the typical experimental
range. CPDs result when the double bond between carbons 5 and 6 of each of two
adjacent pyrimidine rings on the same strand are replaced by single bonds plus two
inter-ring carbon-carbon bonds to form a cyclobutyl ring (most often in the cys-syn
configuration) between the two pyrimidines. Two adjacent thymine (T-T) bases are
most frequently the site of CPD formation (>66% of CPDs), with cytosine-thymine
(C-T) and thymine-cytosine (T-C) sites accounting for the bulk of the remainder
(Friedberg et al., 1995). Sequence context, however, is also known to exert an
influence on the frequency and location of CPD formation (Kunz et al., 1987;
Friedberg et al., 1995). (6-4) photoproducts may also form at T-T sequences, but
more often at T-C and C-C sites (Friedberg et al., 1995). As the name implies, (6-4)
photoproducts are produced by the UV-induced formation of a linkage between the
carbon 6 position of one base and the carbon 4 position of the adjacent base
(Friedberg et al., 1995).
1.1.3 UV-radiation effects on plants
Gross effects of solar UV on plant growth and morphology have been
observed. For example, zooplankton is known to suffer decreased biomass
production under conditions of increased UV-B (Soni and Joshi, 1997). Also,
seedlings of the legume Vigna unguiculata showed increased shoot length, dry and
wet weight, and leaf area under conditions of UV-B exclusion (Lingakumar et al.,
1999). Rice cultivars have been shown to suffer leaf damage in the form of localised
6
necrosis and dessication from exposure to UV-B irradiation (Caasi-Lit et al., 1997).
Incident solar UV-B radiation also is known to effect a number of characteristics of
the summer annual “Chamico” (Datura ferox) (Ballare et al., 1996). When UV-B
was excluded entirely from field plots, seedling emergence during daylight increased
by 70% compared with the control plot, although total seedling emergence for each
24-hour period was not significantly greater. Furthermore, the rate of hypocotyl
elongation was 20-30% greater under UV-B exclusion, and plants not exposed to
UV-B had about half the number of CPD lesions in their genomic DNA as did
exposed plants. These authors also reported that grazing by phytophagous insects on
this species was affected by solar UV-B exposure.
Other authors (Fiscus and Booker, 1995) have contended that the major effect
of UV-B exposure is directly on the proteins or chromophores of photosystem II
rather than at the DNA level (although DNA lesions and particularly CPDs and (6-4)
photoproducts are acknowledged). At a molecular level, UV-B stress applied to
individuals of Pisum sativum caused changes in mRNA and protein levels for sixteen
genes, including increases in the production of phenylpropanoid biosynthesis
pathway proteins (Brosche et al., 1999). Phenylpropanoid derivatives in A. thaliana
and soybean (Glycine max) have since been shown to have a role in protection
against UV, and to be inducible by incident UV-B radiation (Mazza et al., 2000).
1.2 Influence of plant development on DNA repair mechanisms
In contrast to most animal cells, plant cells are totipotent, and do not
developmentally migrate in sheets. The unique way in which plants develop has an
unknown bearing on the requirement for, and mechanisms of, DNA repair. For
example, in animal cancers, it is thought to be an accumulated mutation load that
results in cell proliferation and culminates in a tumor (Loeb, 1991; Loeb and
7
Christians, 1996). This accumulation may be the result of a repair deficiency, or an
overwhelming genotoxic exposure. Such tumorigenic cell proliferation is not
necessarily the case in plants. For example, an increase or decrease in plant cell
number due to alteration of a cyclin concentration had no major effect on the
morphology or function of the plant (Doerner et al., 1996; Doonan and Hunt, 1996),
and certainly did not result in tumorigenic growth. Whether this is an indication that
plants have a less plastic developmental process remains to be seen.
In addition, polyploidy may present an alternative to damage repair in mature
plants, in that multiple copies of each gene may reduce the requirement for repair.
This alternative could be in the form of compensatory expression from a
non-damaged allele, as a result of either a cellular feedback induction/suppression
mechanism, or an alteration of interallelic interactions between all copies of the gene
in question. To render this alternative plausible, all copies of a multicopy gene must
be under such plastic control as to be capable of expression in a particular
circumstance. Evidence supporting this criterion can be found in observations of
hexaploid wheat, and other recent polyploids, which express the majority of the
copies of specific genes (as reviewed by Wendel, 2000). It is also known that
changes in ploidy number can increase or decrease expression levels, as seen in yeast
and maize (Guo et al., 1996; Galitski et al., 1999), further obscuring the requirement
for DNA repair in specific plant species.
The ability of plants to reproduce both sexually and vegetatively, and also to
access continuous meiotic production of gametes must also present alternatives to
molecular repair at a phenotypic selection level. Overall these factors have an
unknown effect on the demands placed on DNA repair systems in plants.
8
1.3 Primary responses to external sources of DNA damage
Organisms exposed to solar radiation often display increased production of
waxy cuticles (Strid et al., 1990), and are typically equipped with UV-absorbing
pigments as primary lines of defense. Various amino acid compounds in
cyanobacteria, and carotenoids, flavonoids or sinapic acid derivatives in plants play
similar shielding roles (Chasan, 1994). The yellow carotenoid pigment in many types
of pollen offers some protection from incident radiation (Jackson, 1987). Flavonoids
such as flavones, isoflavonoids and anthocyanins accumulate in the vacuoles of the
epidermal layer in response to UV-B exposure, (Chasan, 1994; Kootstra, 1994; Lois,
1994; Strid et al., 1994; Takayanagi et al., 1994) and production of protective
terpenoid indole alkaloids is also induced (Ouwerkerk et al., 1999). The effectiveness
of UV-induced phenolics in reducing mesophyllic penetration by solar UV-B has
been shown (Mazza et al., 2000). The importance of these compounds in resistance
to UV-induced damage by plants has been demonstrated using biosynthetic mutants
(Lois and Buchanan, 1994). For example, when flavonoid mutants and wild type
Arabidopsis grown under various UV-B exposures were compared, the growth rate
of the most seriously affected mutant was reduced by approximately 66% (Li et al.,
1993).
1.4 Secondary responses to spontaneous and induced DNA damage
Whereas the primary plant responses to DNA damage and damaging agents
consist largely of protective strategies to minimise the occurrence of damage, the
secondary responses discussed below focus on processing existing damage. Broadly,
these responses can be classified as reversal, in which a single enzyme process
directly reverses a specific type of lesion; repair, in which the lesion is removed
either singly or as a section of DNA; or as tolerance of the damage, in which a
9
replication-blocking lesion is processed in such a way as to allow synthesis past the
damage site. As UV radiation has been used widely as a genotoxic agent in
investigations of the reversal and repair of DNA damage, the following processes are
discussed with particular reference to UV-induced damage.
1.4.1 Photoreactivation
Photoreactivation of DNA damage is a direct reversal process, and is
manifested by the enhanced survival of UV-irradiated organisms concurrently or
subsequently exposed to light (300-500 nm). The process is apparently executed by a
single enzyme, “photolyase” or “photoreactivating enzyme”. The generalised
mechanism of action of this substrate-specific enzyme can be divided into two steps.
Firstly, photolyase binds specifically to DNA containing the target lesion in a
light-independent reaction. This is then followed by the photon-requiring step of
dimer monomerisation, in which the cyclobutane covalent linkages between adjacent
pyrimidine rings are cleaved, thus restoring the original configuration of double
bonds between the fifth and sixth carbons of each pyrimidine ring (Friedberg et al.,
1995).
Photolyases have been identified in numerous prokaryotic organisms, and
some eukaryotes, including plants (Jackson, 1987; Pang and Hays, 1991; Batschauer,
1993; Malloy et al., 1997) but not humans (McLennan, 1988; Friedberg et al., 1995).
Photolyases from prokaryotes have been designated Type I, while CPD photolyases
from higher eukaryotes constitute Type II (Landry et al., 1997). Some organisms
have more than one type of photolyase, each type having a specific substrate
(McLennan, 1988).
In plants, investigations of photoreactivation in Arabidopsis initially revealed
photoreactivating activity (Pang and Hays, 1991), and a cDNA (PHR1) encoding a
10
Type II photolyase specific for CPDs was identified (Taylor et al., 1996; Ahmad et
al., 1997). Reversal of (6-4) photoproducts also has been observed in Arabidopsis
(Chen et al., 1994), culminating in identification of the gene (UVR3) responsible, and
characterisation of an Arabidopsis mutant (see section 1.5) lacking this activity
(Nakajima et al., 1998). Similarly, both CPDs and (6-4) photoproducts are
photoreactivated in wheat (Triticum aestivum) (Taylor et al., 1996), although in other
organisms (6-4) photoproduct photolyases are known to be less efficient in repair
than CPD photolyases (Kim et al., 1994), perhaps reflecting the relative abundance
of the respective lesions.
Four Arabidopsis cDNAs capable of at least partially complementing an
Escherichia coli mutant simultaneously deficient in photoreactivation and two other
repair pathways were isolated. On the basis of phenotypic correction, one of these
clones (DRT103) was suggested to encode a photolyase (Pang et al., 1993a).
However, it showed no amino acid sequence homology with any known DNA repair
protein, and thus its role in photoreactivation remains questionable (the other three
clones, DRT100-102, are discussed below).
The mustard Sinapsis alba yielded a putative photolyase coded by the
saPHR1 gene that was more successful in complementing an E. coli photolyase
mutation (Batschauer, 1993). Amino acid sequence similarity with bacterial and
yeast photolyases was seen, as was induction in leaves by white light, consistent with
the suggested function. Another Arabidopsis cDNA, atPHH1, was isolated by
probing a λYES library with the saPHR1 cDNA probe. The predicted atPHH1
protein showed structural similarities to plant blue-light receptors and microbial
photolyases, and was 89% identical at the protein level with saPHR1. Despite this,
atPHH1 was unable to significantly counteract the UV-sensitivity of a yeast strain
11
(S. cerevisiae) deficient in photoreactivation and other repair pathways (rad2 rad18
phr1) (Hoffman et al., 1996). Hence, the true in vivo function of this protein remains
unknown. Similarities of other photolyase and blue-light receptor genes were
subsequently noted (Ahmad et al., 1997), supporting both the common function of
these two gene products, and also the validity of atPHH1 as a photolyase homologue.
Subsequent investigations revealed the importance of photoreactivation in plants
when it was found that Arabidopsis mutants lacking active photolyases for CPDs or
(6-4) photoproducts are hypersensitive to UV-B (Jiang et al., 1997; Landry et al.,
1997).
1.4.2 Base excision repair (BER)
The excision of misinformational or non-coding bases from eukaryotic DNA
via BER is initiated by DNA glycosylases. The process involves cleavage of the
N-glycosyl bond to liberate a free base and generate an apurinic/apyrimidinic (AP)
site. The AP site is then the substrate for an AP endonuclease which hydrolyses the
phosphodiester bond immediately 5’ or 3’ to the AP site. This nick allows an
exonuclease (DNA deoxyribophosphodiesterase) to remove the terminal
sugar-phosphate moiety in preparation for completion of repair by a DNA
polymerase and DNA ligase 1 (Friedberg et al., 1995). The specific polymerase
concerned is still unknown, however in yeast both polymerase δ and polymerase ε
may be involved in BER (Wang et al., 1993; Blank et al., 1994). An Arabidopsis
DNA ligase 1 (Taylor et al., 1998), which not only showed amino acid sequence
identity of 45% with human DNA ligase 1 (42% with S. cerevisiae ligase 1), but was
able to rescue a temperature-sensitive S. cerevisiae cdc9 (DNA ligase) mutant has
been described.
12
The formation of AP sites has been observed in the DNA of seeds of Zea
mays during early germination (Dandoy et al., 1987). This phenomenon was
attributed to the action of DNA glycosylases on lesions accumulated during seed
storage, implying the presence of BER in this species. Likewise, an enzyme activity
attributed to uracil-DNA glycosylase was found in cultured cells of wild carrot,
Daucus carota (Talpaert-Boyle and Liuzzi, 1982). Repair of 8-oxoguanine (8-oxoG),
an oxidation product of guanine, is effected in E. coli by the action of the mutT
protein, an 8-oxo-dGTPase; or the mutM or mutY DNA glycosylase proteins
(Ohtsubo et al., 1998) that generate a substrate for BER. Homologues of the E. coli
mutM gene (atMMH1 and atMMH2) have been isolated from Arabidopsis, the first
eukaryote to display such conservation (Ohtsubo et al., 1998) (yeasts and mammals
appear to have functional but not structural equivalents of this family of proteins).
The atMMH1 plant protein showed 31% identity and 52% similarity with the E. coli
mutM protein, and furthermore displayed the PELPEV catalytic amino acid motif
essential for the repair activity. This motif is conserved at the amino terminal end of
all mutM homologs. Functional assays in vitro showed that atMMH1 could nick a
double-stranded oligonucleotide adjacent to an 8-oxoguanine when that base was
paired with a cytosine or guanine. The second, longer, atMMH2 cDNA has a
mid-frame insertion of a 158 bp fragment, the sequence of the first 66 nucleotides of
which is very similar to that found downstream in atMMH1, and may represent a
gene duplication and divergence event, coupled with differential RNA processing.
atMMH2 codes for a truncated protein due to the presence of an in-frame stop codon
slightly downstream of the insertion site.
A cDNA encoding 3-methyladenine glycosylase (aMAG) has been isolated
from an Arabidopsis λYES library (Santerre and Britt, 1994). When expressed in an
13
E. coli mutant defective in BER of alkylation damage, this cDNA substantially
increased the resistance of this strain to methyl methanesulfonate. In vitro
biochemical assays of the expressed enzyme confirmed the glycosylase activity.
However, amino acid sequence alignments of aMAG with mammalian
3-methyladenine glycosylases revealed only limited regions of homology with the rat
glycosylase being phylogenetically the closest. In Arabidopsis this gene has been
shown to be expressed in growing tissue (Shi et al., 1997). Interestingly, THI1, a
gene homologous to yeast thiamine biosynthetic genes, also was isolated from
Arabidopsis by complementation of a BER-deficient mutant of E. coli (Machado et
al., 1996). Whether THI1 plays any role in BER in plants remains to be determined.
1.4.3 Nucleotide excision repair (NER)
The NER pathway, first elucidated in E. coli, acts on lesions that have created
significant distortions in the DNA double helical conformation (including CPDs and
(6-4) photoproducts). Initial recognition of a lesion is by a multimeric complex,
ultimately resulting in incision of the damaged strand on both sides of the lesion.
Following, or simultaneously with unwinding of the DNA strands to release the
lesion-containing segment, the gap is filled by a DNA polymerase and then sealed by
a DNA ligase.
1.4.3.1 Human and yeast nucleotide excision repair
In yeast, many components of NER are coded for by genes of the RAD3
epistasis group, and bear “RAD” designations as a result of the radiation sensitivity
usually found in strains carrying mutations in members of this group (Haynes and
Kunz, 1981). Genes and proteins involved in human NER have been studied largely
in conjunction with the clinical condition xeroderma pigmentosum (XP), and
consequently have been designated members of the XP group (de Boer and
14
Hoeijmakers, 2000). Table 1.1 is a listing of genes thought to code for NER proteins
of similar function in several organisms.
1.4.3.1.1 Formation of the repair complex at the lesion
Assembly of the NER complex in yeast and humans is thought to occur in a
sequential manner at the site of the lesion. Previously, XPA/RAD14 was considered
to be responsible for initial damage recognition (Guzder et al., 1993), but recent
progress in defining the function of the XPC•hHR23B complex renders this notion
less likely. In humans, the formation of the XPC•hHR23B complex acts to stimulate
the activity of XPC in sensing and binding to DNA lesions, and thereby recruiting
the remaining repair factors (see below) to the site (de Laat et al., 1999). The precise
role of XPA/RAD14 in NER remains to be elucidated. Perhaps its ability to bind
lesions (Nocentini et al., 1997) is complementary to, or is in addition to, the function
of XPC•hHR23B. Whatever the role of XPA, the yeast RAD14 protein is absolutely
required for NER (Guzder et al., 1995), as is replication protein A (RPA) (de Laat et
al., 1998; de Laat et al., 1999). In fact, in humans RPA stimulates the ability of XPA
to bind UV-induced DNA damage (Stigger et al., 1998). On this basis, an alternative
model of initial lesion recognition and binding in humans specifies formation of an
RPA•XPA•DNA complex at the lesion site (Wakasugi and Sancar, 1999).
The yeast homologue of XPC is RAD4 (Legerski and Peterson, 1992;
Downes et al., 1993), which when mutated results in defective removal of
UV-induced pyrimidine dimers in this organism (Prakash, 1977). RAD23, a yeast
homologue of hHR23B (Friedberg et al., 1995), is required for transcription-coupled
repair (TCR) (Meuller and Smerdon, 1996) (see section 1.4.4), and has been more
extensively studied than RAD4. The yeast RAD23 gene has promoter sequences
similar to the Damage Responsive Element DRE1 and/or DRE2 regions found in
15
other yeast damage-inducible NER genes (Siede et al., 1989) (see below). Mutation
of the yeast RAD23 gene results in moderate UV-sensitivity (Verhage et al., 1996).
Dual mouse RAD23 homologues (van der Spek et al., 1996) and a second human
homologue have been reported (Masutani et al., 1994), and classified into groups ‘A’
and ‘B’. The precise function of group A members is yet to be determined, although
hHR23A appears to have a role in cell cycle progression (Gragerov et al., 1998;
Kumar et al., 1999), and perhaps some interchangeability with hHR23B in the
complex with XPC (Sugasawa et al., 1997). For clarity, a schematic depiction of the
possible arrangement of yeast NER components at a DNA lesion is provided in
Figure 1.1.
1.4.3.1.2 Formation of regions of single-stranded DNA
Interestingly, a number of eukaryotic proteins appear to function in both
transcription and NER (see section 1.1.4). In particular, the yeast and human basal
transcription factor BTF2/TFIIH complex includes RAD3/XPD,
RAD25(SSL2)/XPB, TFB1/P62 and SSL1/P44 (Svejstrup et al., 1995; Araujo and
Wood, 1999; de Laat et al., 1999). In both transcription and repair, the complex is
involved in opening a section of double-stranded DNA helix as a prelude to strand
incision. However, the length of the DNA section melted appears to be different for
the two processes; 17 bp in transcription initiation, and 20-30 bp in NER (de Laat et
al., 1999). Further evidence of linkage between components of NER and
transcription is provided by the finding that both the yeast RAD2 and RAD4 proteins
bind specifically to subunits of TFIIH (Bardwell et al., 1994; Habraken et al., 1996).
For repair, the human TFIIH complex may be recruited to the damage site via
a specific affinity of XPC for the TFIIH subunits XPB and/or p62 (Yokoi et al.,
2000). Once TFIIH is positioned at the damage site, it unwinds the helix through the
16
ATP-dependent action of the XPB and XPD helicases (Araujo and Wood, 1999; de
Laat et al., 1999). In yeast, the RAD25 protein (homologous to the mammalian
XPB/ERCC3 protein) acts as a helicase to construct specific branched DNA
structures at the damage site (Coin et al., 1999; Bradsher et al., 2000). TFIIH also has
some additional, as yet unknown, function specific to NER (de Laat et al., 1999).
1.4.3.1.3 Dual incisions around the lesion
As NER continues, the unwound section of the helix is a substrate for the
structure specific endonucleases RAD2/XPG and RAD10/ERCC1•RAD1/XPF (de
Laat et al., 1999). Incision on the 3’ side of the lesion ‘bubble’ is mediated by
RAD2/XPG (O'Donovan et al., 1994; Mu et al., 1996). The RAD2 family of
nucleases consists of three classes of proteins classified on the basis of sequence
similarity. Each subgroup I, II or III appears simplistically to have a specialist
function in repair, replication or recombination, respectively (Lee and Wilson, 1999).
RAD2, XPG and homologues appear in the Class I group; FEN-1 and its homologues
appear in Class II, and those members displaying a characteristic exonuclease
activity appear in Class III. Members of classes I and II differ in length due to the
absence of a large central part of the class I protein, for example 646 amino acids of
RAD2 are missing from YKL510 (Jacquier et al., 1992; Harrington and Lieber,
1994; Lieber, 1997), the S. cerevisiae FEN-1 homologue. This section will
henceforth focus on RAD2 and its homologues.
The RAD2 endonuclease in yeast is specified by 1,031 codons of which the
last 78 codons of the 3’ end of the gene are required for the RAD2 repair function
(Madura and Prakash, 1986). RAD2 is required for repair of UV-induced damage,
but is not required for viability (Higgins et al., 1984; Naumovski and Friedberg,
1984; Nicolet et al., 1985; Habraken et al., 1993). Both the RAD2 and RAD4
17
proteins bind specifically to subunits of TFIIH (Bardwell et al., 1994; Habraken et
al., 1996; Lyer et al., 1996), implying a link with transcription.
RAD2 (and homologues) may also be involved in BER of alkylation damage
(Cooper and Waters, 1987), and in addition appear to have a structural,
non-endonucleolytic function in NER (Evans et al., 1997; Wakasugi et al., 1997; Mu
et al., 1997b). Two repeated elements in the 5’ flanking regions are common to
RAD1, RAD2 and RAD3 (CS1 and CS2; 146 and 335 bases upstream of the RAD2
START codon, respectively) (Nicolet et al., 1985), possibly representing control
sequences. Evidence points to a lack of cell cycle specific regulation of RAD2 in the
mitotic cell cycle (Madura and Prakash, 1990), and in fact this gene apparently is
constitutively expressed at less than five copies of mRNA per cell in S. cerevisiae
throughout the cell cycle (Siede et al., 1989).
RAD2 is inducible by exposure to DNA damaging agents (Madura and
Prakash, 1986; Siede et al., 1989), and specific promoter elements have been
identified (Siede and Friedberg, 1992). Four cis-acting AT-rich elements (AT1-4),
and two trans-acting regions, named DRE1 and DRE2, were mapped. All elements
act as positive regulators (Siede et al., 1989). DRE1 is located at position -153 to
-168, and DRE2 at -72 to -95, distinct from CS1 and CS2. DRE1 was found to be
required for both induced and constitutive expression of RAD2, and when deleted
resulted in increased UV-sensitivity exclusively in G1/S phase. Deletion of DRE2
abolished inducibility without affecting constitutive expression, and caused increased
UV-sensitivity, which was not cell cycle specific. SNM1, PHR1, RAD23 and RAD7
also have promoter sequences similar to DRE1 and/or DRE2 (Siede and Friedberg,
1992; Wolter et al., 1996). Increased protein binding to both DRE1 and DRE2 was
found on exposure to DNA damaging agents, and was reliant on post-irradiation
18
protein synthesis. All elements required for UV radiation induction of RAD2 were
contained within the 350 bp region upstream of the RAD2 open reading frame (Siede
et al., 1989). The inducible SNM1 gene of S. cerevisiae, involved in the repair of
interstrand cross-links, also has a 25 bp motif in its promoter region which shows
homology to the RAD2 DRE2 box (Wolter et al., 1996).
The human homologue of RAD2 is the XPG gene (ERCC5) (MacInnes et al.,
1993; O'Donovan and Wood, 1993; Scherly et al., 1993; Shiomi et al., 1994). Both
S. cerevisiae RAD2 and human XPG proteins exhibit a 5’→3’ exonuclease activity
(Habraken et al., 1993; Habraken et al., 1994; Habraken et al., 1996). Early
comparison of the nucleotide sequences of RAD2 and related (but not necessarily
homologous) genes in S. pombe (RAD13) (McCready et al., 1989), mice (FEN1) and
humans (XPG/ERCC5) showed 3 conserved regions. These were named the N, I and
C regions (Harrington and Lieber, 1994), and would be expected to appear in other
RAD2 protein homologues. In RAD2 and XPG respectively, these regions
encompass amino acids 1-95 and 1-95 (N region); 756-890 and 752-892 (I region),
and 1011-1031 and unidentified (C region). It has also been established that a
conserved C-terminal region of XPG (amino acids 747-928) interacts directly with
the TFIIH subunits XPB, XPD, p62 and p44, and furthermore, that residues
981-1009 constitute a poliferating cell nuclear antigen (PCNA) binding region (Gary
et al., 1997). Additionally, a conserved N-terminal motif comprised of amino acid
residues 1-377 contributes to the interaction of XPG with only XPB or XPD (Lyer et
al., 1996). Obviously, the XPG C-terminal region required for contact with TFIIH
correlates with the evolutionarily conserved I region, and the XPG N-terminal motif
includes and extends the conserved N region. The stretch of amino acid sequence
missing in Class II homologues (for example FEN-1) is between the N and I regions
19
of RAD2 (Harrington and Lieber, 1994), probably between residues 114 and 752.
This may contribute to the difference between RAD2 and FEN-1 functions in that
FEN-1 does not have part of the N-terminal region thought to interact with XPB and
XPD homologues.
Incision on the 5’ side of the lesion is executed by the
RAD10/ERCC1•RAD1/XPF heterodimer (Bardwell et al., 1994; de Laat et al.,
1998). The yeast RAD1 protein is 1,100 amino acids in length, corresponding to a
cDNA of 3,300 nucleotides. A bipartite nuclear transport motif is assigned to amino
acids 517 onwards (Friedberg et al., 1995), and the RAD10 binding region is thought
to be between amino acids 809 and 997 of RAD1 (Bardwell et al., 1993). In vivo, the
stable RAD1/RAD10 complex displays a magnesium-dependent endonucleolytic
activity on single-stranded DNA substrates (Tomkinson et al., 1993). RAD1 also
appears to play a role in recombination in S. cerevisiae (Kadyk and Hartwell, 1993;
Tomkinson et al., 1993) and, in conjunction with MSH2, in the repair of 26-base
loops (Kirkpatrick and Petes, 1997). The RAD10 protein is much smaller than
RAD1, being 210 amino acids in length. The RAD1 binding region is located within
a hydrophobic region of RAD10 bounded by amino acids 90 and 210 (Bardwell et
al., 1993). XPA is able to bind ERCC1, via a binding region in that protein distinct
from that involved in the ERCC1-XPF interaction (Li et al., 1994; Park and Sancar,
1994). Whether the yeast homologues of XPA and ERCC1 (RAD14 and RAD10,
respectively) also have a direct interaction is unclear (Li et al., 1994). However,
human RPA is able to directly bind the ERCC1 and XPG proteins (Park and Sancar,
1994; He et al., 1995; de Laat et al., 1998; Araki et al., 2000), prompting the
suggestion that RPA may also play a role in the inhibition of DNA replication in the
presence of DNA damage (Wang et al., 1999).
20
Dual incisions allow the removal of the damaged section of DNA followed
by repair synthesis. Apparently either or both polymerase (Pol) δ and Pol ε are able
to participate in repair synthesis (Budd and Campbell, 1995; Budd and Campbell,
1997; Wood and Shivji, 1997) in conjunction with PCNA (Shivji et al., 1992). DNA
backbone breaks are then sealed, again, probably by DNA ligase 1 (Friedberg et al.,
1995).
1.4.3.2 NER proteins and cell cycle progression in humans
The p53 protein is a major regulator of human cell cycle progression,
including the maintenance of genomic stability through coordination of cell division,
DNA replication and DNA repair (Kastan et al., 1991; Marx, 1993; Marx, 1994;
Pellegata et al., 1996; Stillman, 1996; Levine, 1997).
Human XPB and XPD both bind to wild-type p53 in vitro (and in vivo for at
least XPB), thereby providing such a link between NER and cell cycle progression in
humans (Wang et al., 1994; Wang et al., 1995). This link is necessary to allow
temporal organisation of these processes. Furthermore, in vivo binding of TFIIH by
wild-type p53 can result in reduced TFIIH helicase activity. This interaction
appeared to be localised to the carboxy-terminal domain (CTD) of p53, specifically
residues 367-393 (Wang et al., 1995). Several p53 mutants failed to exhibit the same
inhibitory effects. It is also known that the p53-mediated apoptosis pathway is
deficient in XPB or XPD mutant cell lines (Wang et al., 1996), consistent with direct
binding of the CTD of p53 by XPB, and an unknown interaction with XPD also
involving the CTD of p53. This interaction may be the linkage between
repair/transcription and cell cycle progression. Details regarding the specific location
of the p53 binding site on XPD are not available, however the analogous relationship
between p53 and XPB is thought to involve the third helicase domain of XPB (Wang
21
et al., 1995). This binding of XPB/ERCC3 was almost completely inhibited by the
human Hepatitus B x (HBx) protein (Wang et al., 1994), and it is also known that
HBx inhibits NER generally as indicated by sensitivity to UV-C (Jia et al., 1999).
Given that the helicase activity of TFIIH is dependent on XPB rather than XPD
(Coin et al., 1999), the above differences in in vitro and in vivo binding by p53 may
be quite significant, and in fact the ability of p53 to modulate the activity of TFIIH
may well depend in vivo on binding to XPB alone. In addition, human p53 as a
homotrimer is also able to bind directly to DNA damage in the form of
insertion/deletion mismatches (Lee et al., 1995).
1.4.3.3 NER proteins and transcription-coupled repair
In bacteria, yeast and humans there is a bias towards NER of actively
transcribed genes, usually manifested as preferential repair of the transcribed strand
(Bootsma and Hoeijmakers, 1993; Selby and Sancar, 1993; Friedberg et al., 1995;
Mellon et al., 1996a). Eukaryotic NER also may be coupled with other processes
involving disassembly or reassembly of the nucleosome (Roberts, 1988; Bootsma
and Hoeijmakers, 1993; Feaver et al., 1993; Selby and Sancar, 1993; Friedberg et al.,
1995; Gaillard et al., 1996; Mellon et al., 1996a). In the yeast S. cerevisiae, RAD23
is required for TCR (Meuller and Smerdon, 1996), and apparently promotes complex
formation between TFIIH and the RAD14 protein. RAD4/XPC is dispensable for
TCR (de Laat et al., 1999), perhaps because lesion recognition in this situation may
be via a stalled RNA polymerase II complex (Donahue et al., 1994). Interestingly,
the stalled polymerase complex has a footprint of about 40 nucleotides symmetrically
arranged around the lesion (Selby et al., 1997), and results in inhibition of
photoreactivation of the lesion (Donahue et al., 1994). In vitro observations with
yeast (You et al., 1998) suggest that when the processes of transcription and NER
22
compete, the TFIIH complex is preferentially sequestered for NER resulting in
inhibition of transcription. This preferential mobilisation of dual function complexes
for NER was not seen in a rad26 mutant, suggesting a pivotal role for RAD26 in
regulation of transcription, NER, or both. Consistent with this, and further evidence
of the duality of these proteins in both transcription and NER, TCR is not seen in
rad26 mutants (Tijsterman and Brouwer, 1999). In fact, removal of CPDs from the
transcribed strand is slowed, perhaps in accordance with an inability to process
stalled RNA polymerase II complexes.
1.4.3.4 Plant nucleotide excision repair
Few investigations of NER in plants have been reported and the results have
often been in conflict. The rate of photoreactivation increases in alfalfa roughly as a
linear function of the CPD frequency, until a maximum is reached, apparently
reflecting saturation of the photoreactivation pathway. Increases in the rate of repair
beyond this point have been attributed to NER (Quaite et al., 1994a; Quaite et al.,
1994b). NER also was suggested to occur in soybean (Glycine max) and cultivars of
rice (Oryza sativa) based on the reduction of nuclear CPD frequency (Cannon et al.,
1995; Sutherland et al., 1996; Hidema et al., 1997). Furthermore, CPDs were found
to be excised from the nuclear DNA of Daucus carota and Wolffia microscopia at
rates dependent on damage levels and comparable to those in animal cells (Degani et
al., 1980; Eastwood and McLennan, 1985; McLennan, 1987; Hidema et al., 1997).
Consistent with the foregoing, a UV-specific endonuclease resembling UvrABC
nuclease in activity was partially characterised from spinach (Doetsch et al., 1989).
In contrast, studies of photoreactivation in Arabidopsis indicated that the removal of
CPDs in the dark proceeded at a rate about one order of magnitude lower than that of
photoreactivation (Pang and Hays, 1991). Indeed, little or no dark repair of CPDs
23
was observed in either the nuclear or organellar DNA of Arabidopsis (Britt et al.,
1993; Chen et al., 1996). Similarly, an examination of UV damage repair in soybean
chloroplasts revealed the presence of a slow, light-dependent process, but no dark
repair (Cannon et al., 1995). On the other hand, repair of (6-4) photoproducts in the
absence of light has been reported for Arabidopsis; and the UV-sensitivity of an
Arabidopsis mutant was determined to be due to defective dark repair of (6-4)
photoproducts (Britt et al., 1993). Recently, UV-B sensitive rice cultivars were
shown to be deficient in both photorepair and NER as a consequence of polygenic
mutations (Hidema et al., 1997).
Two Arabidopsis cDNAs, DRT101 and DRT102, were isolated on the basis of
the ability to partially complement the UV-sensitivity of an E. coli mutant, and were
suggested to encode UV-specific excision repair enzymes (Pang et al., 1993a). Of the
two, DRT101 also appeared to be DNA damage inducible. However, additional
analysis revealed that the predicted DRT101 protein includes an N-terminal
chloroplast transit peptide and that neither of the predicted proteins featured any
significant homology with known DNA repair enzymes.
Since the initiation of this study, other evidence contributing to the definition
of NER in plants has been published. A Rad25/XPB homologue was retrieved from
Arabidopsis by a combination of degenerate PCR and cDNA library screening
(Ribiero et al., 1998), and named XPBara by these authors. In yeast, RAD25 is a
component of the TFIIH transcription factor, and possesses an ATP-dependent
helicase activity (Guzder et al., 1994), and is involved in the excision step of NER
(Sung et al., 1996). In humans, mutation of this gene leads phenotypically to
xeroderma pigmentosum Group B (XP-B) (Weeda et al., 1990). The Arabidopsis
protein showed 48% and 53% identity and 70% and 73% similarity with the yeast
24
and human proteins respectively. Amino acid conservation was noticeably higher in
domains known to be functionally required in the yeast or human proteins (including
the putative p53 binding site). Despite this similarity, the plant cDNA was not able to
complement for UV-sensitivity in XPB-deficient CHO cells (Ribiero et al., 1998).
Recently, the sequence and expression data for a rice RAD23 structural
homologue (OsRAD23A), discovered as a result of screening to identify proteins
involved in the control of transcription, has been reported (Schultz and Quatrano,
1997). As discussed in section 1.4.3.1.1 above, yeast RAD23 is involved in the NER
pathway, and is required for TCR (Meuller and Smerdon, 1996). Mutation of the
yeast RAD23 gene results in moderate UV-sensitivity, probably as a result of a
general NER defect (Verhage et al., 1996). OsRad23A shows about 50% sequence
similarity with the corresponding human, mouse and yeast RAD23 proteins. The
function of this gene in NER in rice, if any, has not been determined. This report also
indicated the presence of several putative RAD23 homologues in the Arabidopsis
EST databases.
A monocot (Lilium longiflorum) partial cDNA prepared from generative cells
showed 52% identity (71% similarity) to human ERCC1, and 30% identity (39%
similarity) to yeast RAD10 (Xu et al., 1998). Functionally, the lily cDNA was able to
complement ERCC1-deficient Chinese hamster ovary cells for sensitivity to
mitomycin C (a recombinational repair pathway), but unable to restore, or even
significantly improve, UV resistance. This was attributed to differences in the
quantitative requirements of the recombinational repair vs NER pathways rather than
conservation of particular domains in the lily ERCC1 protein, given that the
uncharacterised region of the truncated lily cDNA was dispensable for DNA repair
by the human ERCC1 protein (Sijbers et al., 1996). It is relevant that this cDNA was
25
retrieved by virtue of its corresponding mRNA being up-regulated specifically in
generative cells isolated from pollen. As discussed above in section 1.1.1,
circumstantial evidence for the existence of NER came primarily from studies of
germinating seeds and, to a lesser extent, pollen.
During the course of this study, an XPF homologue sequence from
Arabidopsis (UVH1) was submitted to the Genbank database (Liu et al., 1999).
Further details of the UVH1 mutation, and also of an Arabidopsis RAD1 homologue
(atRAD1) became available immediately prior to the submission of this thesis, in
independent publications from two separate laboratories (Gallego et al., 2000; Liu et
al., 2000). The results reported in these publications are discussed in section 4.3
below.
1.4.4 Mismatch correction
Systems that post-replicatively repair DNA replication errors (and also act on
certain types of DNA damage that resemble base mismatches) have been identified
in E. coli, yeast and human cells. General mismatch repair in E. coli requires the
mutS, mutH, mutL and uvrD genes, as well as specific exonucleases, DNA
polymerase III and DNA ligase (Friedberg et al., 1995). mutS protein binds
preferentially to DNA containing mismatches, and mutL subsequently binds to the
mutS/mismatch complex. mutH is a mutS/mismatch-dependent endonuclease
(Wagner and Radman, 1995) that generates single-stranded nicks at 5’-GATC-3’
sequences in the undermethylated strand of hemimethylated duplex DNA, thereby
providing strand discrimination. A segment of single-stranded DNA containing the
incorrect base is removed by concerted uvrD and exonuclease activity and the gap is
patched by DNA polymerase and DNA ligase.
26
Homologues of the E. coli mutS and mutL genes (but not mutH) have been
identified in yeast and human cells, although the number of genes involved in
eukaryotes is larger (e.g. MSH1-MSH6, PMS1-PMS6 and MLH1-MLH4 in yeast)
(Reenan and Kolodner, 1992; Prolla et al., 1994b; Crouse, 1996). The presence of
multiple homologues of mutS and mutL presumably points to the complexity of the
eukaryotic mechanism of action, but also may reflect potential roles for certain of
these proteins in processes other than mismatch repair. For example, MSH4 shows
extensive amino acid sequence homology to bacterial mutS protein but appears to act
in recombinational repair rather than mismatch correction (Ross Macdonald and
Roeder, 1994). It is also known that bacterial mutS and mutL are required for TCR
(Mellon and Champe, 1996b).
An Arabidopsis homologue of MSH2 (atMSH2) has recently been isolated
(Culligan and Hays, 1997) by probing cDNA and genomic libraries. Probes were
generated by PCR of genomic DNA or cDNA using degenerate primers
corresponding to conserved regions of mutS/MSH2 genes. Phylogenetic analysis
indicated that the plant gene was most closely related to yeast MSH2 and most
distantly related to bacterial mutS, showing 37% identity at the amino acid level to
the yeast and human proteins. These same authors reported multiple clones
predicting a protein resembling eukaryotic MSH6 (Culligan and Hays, 1997;
Culligan and Hays, 2000). The presence of atMSH2 and atMSH6 was the first
confirmation of the existence of a mismatch correction system in plants. Subsequent
reports described isolation of atMSH3 and two atMSH6 homologues (Ade et al.,
1999), and a mutL homologue (atMLH1) from Arabidopsis chromosome 4 (Jean et
al., 1999). The atMUTL protein showed at least 47% (395 of 843) amino acid
identity with the human, rat, Drosophila or yeast proteins. No complementation
27
studies involving MLH1 mutants of other organisms was undertaken. However the
characteristically increased microsatellite instability was not seen in an Arabidopsis
line carrying a T-DNA insertion in the promoter region of the atMLH1 gene,
although this insertion did not significantly alter the expression of atMLH1. The
region of the plant protein corresponding to the suggested PMS1-binding domain of
the MLH1 protein shows 24% (60 of 255) identical amino acids. The ‘GFRGEAL’
amino acid motif is conserved in all known MLH1 homologues, including this one.
Other motifs are less well conserved in the plant protein. Concomitant screening of
the Genbank database resulted in putative atPMS1 and atMLHx sequences, which
were not further examined.
It was anticipated that this system would exhibit the complexity so far
observed in other eukaryotes, and indeed recent in vitro data supported this notion
when it was revealed that atMSH2 forms a heterodimer with atMSH3 capable of
binding to a 1 or 3 base insertion/deletion mismatch. Similarly, atMSH2 and atMSH6
together showed strong selective binding to a single base insertion/deletion, and
atMSH2 in complex with the novel atMSH7 showed moderate specificity for a T•G
mispair (Culligan and Hays, 2000).
1.4.5 DNA damage tolerance
UV-induced DNA damage that is not removed prior to DNA replication in
the bacterium E. coli may be tolerated by recA-dependent translesion synthesis
(TLS), which produces mutations, or error-free post-replication mechanisms that, via
recombination, fill nascent strand gaps left opposite the lesions during replicative
bypass. Subsequently, the damage may be removed via NER (Friedberg et al., 1995).
Damage tolerance mechanisms also appear to exist in S. cerevisiae and
depend on genes within the RAD6 epistasis group for the repair of UV damage
28
(Lawrence, 1994; Friedberg et al., 1995; Nickoloff and Hoekstra, 1998). Members of
this group function in the replication of damaged DNA. RAD6 encodes an
ubiquitin-conjugating enzyme that forms a complex in vivo with the product of the
RAD18 gene, a DNA binding protein (Bailly et al., 1994; Bailly et al., 1997). RAD6
and RAD18 are required for both modes of damage tolerance as determined by their
epistatic relationships to other members of the group, including RAD5, RAD30,
MMS2, REV1, REV3, and REV7 (Johnson et al., 1992; McDonald et al., 1997;
Broomfield et al., 1998), and their effects on damage-induced lethality and
mutagenesis (Lawrence, 1981; Kunz et al., 1992; Armstrong et al., 1994). It has been
suggested, therefore, that a RAD6-RAD18 complex may recognise single-stranded
DNA generated when DNA polymerase stalls at damage sites and, by ubiquitinating
target proteins, promote damage tolerance (Bailly et al., 1997).
There are human homologues of some of the RAD6 group members
suggesting that the same tolerance mechanisms may be active in all eukaryotes.
However, these tolerance mechanisms appear to differ somewhat from those in
prokaryotes. As yet, there is no evidence for post-replication filling of gaps in the
nascent strand by recombination with a sister chromatid. Indeed, gaps may not be
formed when the damage is in the leading strand template, and replicative bypass of
UV lesions in yeast, simian and rodent cells may not involve strand exchange
(Sarasin and Hanawalt, 1980; Resnick et al., 1981; Spivak and Hanawalt, 1992;
Cordeiro-Stone et al., 1999). Similarly, 'template switching' during replicative DNA
synthesis to circumvent a replication block (Higgins et al., 1976) and prevent gap
formation has not been demonstrated. Instead, recent evidence (Svoboda and Vos,
1995; Cordeiro-Stone et al., 1997; Cordeiro-Stone et al., 1999; Masutani et al., 1999)
suggests that a lesion in the lagging strand template may be tolerated by TLS without
29
gap formation. Alternatively, replication may terminate at the lesion, synthesis may
continue at the next Okazaki fragment resulting in a gap opposite the lesion, and
post-replicative TLS may fill the gap. Tolerance of a lesion in the leading strand
template may involve TLS following uncoupling of leading and lagging strand
synthesis. Thus, eukaryotic cells may rely primarily on TLS for replicative bypass of
DNA lesions.
1.4.5.1 Translesion synthesis in yeast and humans
Tolerance of unrepaired DNA damage via TLS allows survival of the cell, but
perhaps at the cost of some replicative fidelity. Subsequently, the damage may be
removed via photoreactivation or NER (Friedberg et al., 1995). Parallel ‘tolerance’
mechanisms appear to exist in S. cerevisiae and involve members of the RAD6
epistasis group for either RAD30- or REV3-dependent bypass of the lesion (for a
review see Kunz et al., 2000).
DNA polymerase η (Pol η) was first detected in yeast where it is encoded by
RAD30, a UV-inducible gene (in exponential phase cells) the deletion of which
confers modest UV sensitivity in stationary and exponential phase cells (McDonald
et al., 1997; Roush et al., 1998; Johnson et al., 1999a; Johnson et al., 1999b). In vitro,
this polymerase efficiently replicates past the TT CPD, usually inserting adenine
opposite each thymine (Johnson et al., 1999b). Estimates of replicative fidelity of
human Pol η suggest that the average error rate for misincorporation of
deoxynucleotides is on the order of 10-2 to 10-3, on both TT-CPD-containing and
undamaged templates (Johnson et al., 2000). Significantly, this fidelity is about 100
times less than that of Pol δ. These observations suggest that Pol η-mediated TLS is
induced in response to UV damage, and contributes to UV resistance by providing a
template for subsequent NER of the TT-CPD damage.
30
Human Pol η was discovered as a consequence of investigations of the
variant form of xeroderma pigmentosum (XPV), an autosomally inherited genetic
disorder that sensitises cells to UV radiation. XPV fibroblasts exhibit normal NER
but are slightly UV-sensitive because of a defect involving diminished TLS past (at
least) TT-CPDs on the leading and lagging strand templates (Cordeiro-Stone et al.,
1997; Ensch-Simon et al., 1998; Svoboda et al., 1998; Masutani et al., 1999). A DNA
polymerase that complements XPV was shown to synthesise past the TT-CPD and
incorporate adenine (Masutani et al., 1999). This reaction was not enhanced by
PCNA, and the polymerase appeared to lack proofreading activity. Subsequent
studies indicated that the polymerase is a human homologue of yeast Pol η, and that
most XPV cell lines tested have inactivating mutations in the Pol η gene
(hRAD30A/XPV) (Masutani et al., 1999; Johnson et al., 1999c).
In vitro studies of the activity of yeast Pol η on damage other than TT-CPDs
have indicated that TLS by this polymerase may not be restricted to processing of
UV photoproducts. The ability of Pol η to insert a base opposite an abasic site
inherently confers an observed mutagenic component to the TLS activity of this
polymerase. Interestingly, although Pol η could not extend the nascent strand beyond
this insertion, DNA polymerase ζ (see below) could subsequently extend from the
Pol η-inserted base to the end of the template, thereby effecting TLS (Yuan et al.,
2000).
The yeast REV3 and REV7 genes encode subunits of the dimeric DNA
polymerase ζ (Pol ζ) also thought to be capable of TLS (Nelson et al., 1996a). The
inactivation of REV3 confers an antimutator phenotype (Cassier et al., 1980; Quah et
al., 1980; Roche et al., 1994), consistent with a mutagenic tolerance process in
wild-type cells. The rev3 antimutator has relatively broad specificity affecting all
31
base-pair substitutions as well as single base-pair deletions/insertions (Kunz et al.,
1998). Indeed, the magnitude of the antimutator effect suggests that at least 60% of
the spontaneous single base-pair substitutions and deletions might be due to
mutagenic translesion synthesis. If so, Pol ζ might be involved in processing many
different lesions, and therefore a substantial fraction of mutagenesis in yeast might
reflect the activity of error-prone mechanisms for tolerance of DNA damage. The
fact that the rev3 antimutator counteracts several NER rad mutators, although to
different extents (Roche et al., 1995), implies that the relevant DNA lesions normally
are substrates for error-free nucleotide excision repair.
Translesion synthesis may be a fundamental mechanism largely independent
of the phylum of an organism, and in fact may even be a primary source of diversity.
The ability to tolerate damage by TLS allows an individual cell to circumvent a
potentially lethal replication block. Error-prone TLS carries an increased risk of
mutation but in single-celled organisms, this may be an attractive compromise given
the need to rapidly adapt to environmental changes. In multicellular organisms that
maintain stable internal environments, however, the increased risk of mutation
accompanying error-prone TLS may seem to be less attractive than the loss of an
individual cell due to replication-blocking damage. For example, the apparent
inactivation of TLS by Pol η in XPV cells leads to enhanced UV mutagenesis (which
seems to involve Pol ξ) and to skin cancer in individuals afflicted with this condition.
Where the generation of diversity is of increased benefit to a multicellular organism,
as in the case of immunoglobulin production (Neuberger and Milstein, 1997; Winter
and Gearhart, 1998), an exception to this rationale may arise. Nonetheless, if the
benefits of error-prone TLS are questionable in multicellular organisms, is Pol ξ a
liability? It has been speculated that diminishing Pol ξ activity in humans by
32
therapeutic intervention, or circumventing it by enhancing Pol η activity, might
decrease the risk of damage-induced mutagenesis and so cancer (Gibbs et al., 1998;
McDonald et al., 1999; Johnson et al., 1999c). If true, this may also apply to other
multicellular eukaryotes.
1.4.5.2 DNA damage tolerance in plants
As detailed above, the phenotypic consequences of translesion synthesis have
been well-documented in S. cerevisiae, and the fundamental genetics of the process
have been derived, yielding a good model upon which to base a parallel investigation
in higher plants. Although there is no direct evidence for translesion synthesis in
plants, the reduction of UV mutagenesis in maize pollen by photoreactivation
(Ikenaga and Mabuchi, 1966) is consistent with the existence of translesion
synthesis. No plant gene encoding a DNA polymerase involved in TLS has yet been
published, nor appeared in a database. However, given the apparent ubiquity of TLS,
there is every likelihood of the existence of such a system in plants. In support of this
possibility, genes encoding homologues of the S. cerevisiae RAD6 protein have been
isolated from wheat (UBC1) and Arabidopsis (atUBC1, atUBC2, atUBC3) using
immunological or polymerase chain reaction (PCR) based approaches (Sullivan and
Vierstra, 1991; Sullivan et al., 1994; Zwirn et al., 1997). atUBC1-atUBC3 exhibit
83-99% identity with each other and the proteins they are predicted to encode show
63-65% identity with the yeast RAD6 protein and are 75% identical to the human
RAD6 equivalent. When expressed in rad6 mutants, atUBC2 but not atUBC1 was
able to partially complement the UV sensitivity of the yeast strain (Sullivan and
Vierstra, 1991; Zwirn et al., 1997); the reason for this difference is not clear as the
atUBC1 result was reported as an unpublished observation.
33
1.4.6 Recombinational repair
In E. coli recombinational repair is largely dependent on the recA gene
whereas members of the RAD52 epistasis group for resistance to ionising radiation
have been implicated in recombination-mediated double-strand break repair in
S. cerevisiae (Friedberg et al., 1995). A damage-inducible Arabidopsis cDNA
(DRT100) that restored some UV resistance to a UV-sensitive E. coli triple mutant
was able to partially complement recA phenotypes associated with recombination
deficiencies (Pang et al., 1992). However, the predicted protein exhibited little
homology to bacterial RecA proteins and contained a chloroplast transit peptide. On
the other hand, an Arabidopsis cDNA isolated via hybridisation to part of a
cyanobacterial RecA gene showed 61% homology to the cyanobacterial RecA
protein, and was cross-reactive with antibodies to the E. coli RecA protein (Cerutti et
al., 1992; Binet et al., 1993). The predicted plant protein also appeared to be
chloroplast targeted. Two additional Arabidopsis cDNAs (DRT111 and DRT112) for
genes potentially involved in homologous recombination were isolated by selecting
for increased resistance of an E. coli ruvC recG double mutant to methyl
methanesulfonate (Pang et al., 1993b). Although neither protein bore any global
similarity to any known repair protein, the DRT111 amino acid sequence contained a
DNA binding motif detected in many DNA repair and recombination proteins, and
the predicted DRT112 polypeptide exhibits 75% identity to Arabidopsis
plastocyanin. Further complementation studies in the relevant E. coli mutants
revealed an increased resistance to UV and greater proficiency in the resolution of
recombination intermediates. Once again, both predicted proteins contained
sequences characteristic of chloroplast targeting domains. Thus, although the DRT
34
gene products may participate in repair and/or recombination in Arabidopsis, the data
argue against a role for these four DRT alleles in the repair of nuclear DNA.
Arabidopsis mutants deficient in homologous recombination are known,
however, one of which in particular (xrs9) phenotypically resembles yeast mutants of
the RAD52 epistasis group, in that it is sensitive to x-rays and methyl
methanesulfonate (Masson and Paszowski, 1997). The specific sequence change
responsible for production of this mutant has not yet been reported. RAD51
(atRAD51) and DMC1 (atDMC1) homologues have been isolated from Arabidopsis
and shown to be expressed in meiotic and/or mitotic tissues (Doutriaux et al., 1998).
Expression of atRAD51 is induced by treatment with gamma (Doutriaux et al., 1998)
and UV-B irradiation (Ries et al., 2000), atDMC1 is not (Doutriaux et al., 1998);
both genes appear to be cell cycle regulated.
A mutation at the A. thaliana MIM locus on chromosome 5 which bestows
sensitivity to X-rays, UV-C irradiation and mitomycin C also has been characterised
(Mengiste et al., 1999). The predicted wild-type protein sequence shows domain
similarity to the SMC (structural maintenance of chromosomes) family of proteins
from S. cerevisiae, and RAD18 from S. pombe compared to which it has an overall
amino acid identity of 26.8%. The S. pombe RAD18 protein is an essential protein
thought to act in some alternative repair pathway providing protection from both UV
and γ radiation, and also possibly have some role in DNA replication and
recombination (Lehmann, 1995). In fact mim mutants were also shown to display a
reduced frequency of intrachromosomal recombination events. This, in conjunction
with the sensitivity to DNA damaging agents led to the suggestion that the MIM
protein functions in an homologous recombinational repair pathway in Arabidopsis,
35
perhaps in creating chromatin structural features required for presentation to, or
recognition by, repair proteins (Mengiste et al., 1999).
1.5 Radiation-sensitive Arabidopsis mutants
The isolation of mutants hypersensitive to DNA-damaging agents has
contributed to our understanding of the mechanisms of DNA repair in both
eukaryotes and prokaryotes. In plants this approach has resulted in several
collections of radiation-sensitive mutants of Arabidopsis. Table 1.2 is a listing of
plant repair mutants, and specifies the original progenitor line, the sensitivity to both
UV and ionising radiation, and tissue-specific sensitivity to these agents. These
mutants were generated by ethylmethanesulfonate mutagenesis of the Colombia or
Landsberg erecta ecotypes (Feldmann et al., 1994) or T-DNA insertional
mutagenesis of the Wassilewskija ecotype (Feldmann and Marks, 1987).
Mutants were selected primarily by the amount of leaf curling/yellowing they
displayed in response to a controlled UV-B or UV-C exposure as compared to
wild-type (with the exception of the A. thaliana rad1-rad5 mutants which were
screened for γ radiation sensitivity (Jenkins et al., 1995)). Secondary screenings
consisted generally of the estimation of growth inhibition compared to wild-type of
either the seedling or root following treatment with UV-B or UV-C. The radiation
sensitivity symbol allocated to each mutant for each test in Table 1.2 often represents
a summation of the results for several exposure intensities for that mutant, and so
carries a significant experimental uncertainty. Therefore, this sensitivity notation
should be taken only as a general guide. The uncertainty highlights the difficulties
involved in identification and characterisation of these putative repair mutants, and
36
may account for the apparent UV-insensitivity of certain seedlings or roots under
certain test conditions.
Some of the mutants have been characterised to distinguish shielding defects
from possible DNA repair defects. This was achieved primarily by quantifying the
formation and removal of UV-induced lesions through the use of immunological
techniques based on antibodies specific for particular UV photoproducts. Defects in
shielding also can be detected by chromatographic analysis of pigment levels. The
comparison between UV sensitivities under photoreactivating or
non-photoreactivating conditions allows defects in photolyases to be distinguished
from defective dark repair mechanisms. Considerations of the DNA repair
mechanism(s) affected in many of these mutants have been based on comparisons
with repair-deficient S. cerevisiae mutants and so must be interpreted cautiously.
The rad1-5 mutants (Davies et al., 1994), are extremely sensitive to γ
radiation but appear insensitive to UV radiation, although only the response of the
plant leaves to UV-C has been tested. This pattern is similar to that observed for S.
cerevisiae mutants assigned to the RAD52 epistasis group (Friedberg et al., 1995).
Members of this group are markedly sensitive to ionising radiation but only slightly,
or not sensitive, to UV radiation, and are required for recombinational repair of
double-strand DNA (dsDNA) breaks.
The uvh1 and uvh3 mutants (Harlow et al., 1994; Jenkins et al., 1995) are
very sensitive to both ionising and UV radiation, reminiscent of the S. cerevisiae
RAD6 epistasis group members (e.g., rad6, rad18) thought to be involved in
post-replication repair (Friedberg et al., 1995). Other mutants (uvh4, uvh5, uvh6)
were also reported from the same laboratory (Jenkins et al., 1995). All are
UV-sensitive (uvh4 and uvh5 only moderately so; uvh6 is hypersensitive), and
37
competent in photoreactivation, whilst uvh4, uvh5, and uvh6 are moderately sensitive
to ionising radiation. uvh1, uvh3, uvh6 were found to be located on chromosomes 3,
3, and 5 respectively. The phenotype of uvh5 also resembles more the rev mutants of
the RAD6 epistasis group (see section 1.4.6). Epistasis analysis in the form of
outcrosses followed by UV challenge indicated that each mutant belongs to a
separate complementation group. uvh1 and uvh3 were suggested to carry a
recombination defect; uvh4 and uvh5 a leaf-specific repair deficiency; and uvh6 a
chloroplast defect. However, since transgenes are integrated into the host genome by
either homologous or illegitimate pathways, inability for transgene integration would
be expected from a mutant with a recombinational defect. Subsequent investigations
of the ability of the uvh1 mutant to integrate a T-DNA transgene revealed an
essentially wild-type response (Preuss et al., 1999). In fact, as discussed in section
1.4.3.4, the mutation causing the uvh1 phenotype has recently been characterised as a
nucleotide substitution in the Arabidopsis homologue of the yeast RAD1 gene.
uvr1, uvr5 and uvr7 (Britt et al., 1993; Jiang et al., 1997) are deficient in the
repair of (6-4) photoproducts in the dark, and so were suggested to be defective in
NER. However, all three also are sensitive to ionising radiation unlike bona fide
NER mutants from other organisms. Nonetheless, NER does have a broad substrate
specificity and so may be involved in the repair of one or more rare γ-induced DNA
lesions in Arabidopsis (Jiang et al., 1997). Complementation studies have shown that
uvh3 is allelic with uvr1 (Jiang et al., 1997) which further complicates identification
of the DNA repair mechanism(s) affected in these mutants.
A number of the mutants apparently defective in certain types of
photoreactivation have been isolated. uvr2 and uvr2-1 (each recovered by a different
laboratory) are impaired in the removal of CPDs under photoreactivating conditions,
38
both map to the same location and have a nonsense mutation in the PHR1 gene
indicating that they probably are allelic, and uvr2-1 has no CPD photolyase activity
in vivo (Jiang et al., 1997; Landry et al., 1997). It has been noted, however, that the
PHR1 gene may only encode a blue light receptor that regulates the actual photolyase
gene (Jiang et al., 1997). uvr3 was shown to be defective in the elimination of (6-4)
photoproducts, but not CPDs, under photoreactivating conditions. However, (6-4)
photoproducts were removed under non-photoreactivating conditions (Jiang et al.,
1997). The mutated gene (UVR3) subsequently found to be responsible for the uvr3
phenotype in fact codes for a class 1 (6-4) photolyase. This protein shows 45-50%
identity with Drosophila and Xenopus homologues, and a human protein of unknown
function (hsCRY1), and was able to partly functionally complement a uvrA-, recA-,
phr- repair-deficient E. coli strain (Nakajima et al., 1998). uvs1-uvs11 also may be
defective in photoreactivation as they are UV-sensitive under photoreactivating
conditions but have wildtype levels of resistance under non-photoreactivating
conditions (Chatterjee, 1997). However, the issue is clouded by the finding that six
of the eleven mutants also are sensitive to ionising radiation.
The remaining 42 uvs mutants are sensitive to UV under
non-photoreactivating conditions only, and so possibly harbour defects in dark repair
mechanisms. In addition, 27 of the 42 isolates appear to be sensitive to γ radiation.
Table 1.2 also illustrates the problems that are now arising in regards to
nomenclature. The use of the designations rad1-rad4 for γ radiation-sensitive
mutants of Arabidopsis can cause confusion as such names have long been used to
describe mutant alleles of the S. cerevisiae RAD3 epistasis group whose members
function in NER. In addition, certain mutants isolated independently appear to have
defects within the same gene, as revealed by complementation studies (uvr1 and
39
uvh3, uvh1-1 and uvh1-2 (Jiang et al., 1997)) or molecular analysis and genetic
mapping (uvr2 and uvr2-1 (Landry et al., 1997)). Such difficulties illustrate the need
for a coordinated series of complementation tests and a standardised system of
nomenclature for Arabidopsis mutants (Meinke and Koornneef, 1997).
1.6 A. thaliana as an experimental organism
A. thaliana is a relatively small dicotyledonous angiosperm. Within the
variability of the different ecotypes, it grows to approximately 20 cm in height over a
period of 5 to 8 weeks. Seeds are set at about 6 weeks after germination, and can
retain some viability after harvest for several years. The diploid genome is comprised
of about 1-1.2 x 108 basepairs, one of the smallest genomes known in angiosperms,
carried in 5 chromosomes ranging between about 13 and 25 Mb (Meyerowitz, 1994).
It has relatively little repeated sequence or inverted repeat sequence and is able to be
completely represented by only 16,000 different lambda library clones (Meyerowitz
and Pruitt, 1985; Meinke et al., 1998). It is estimated that more than half of the
Arabidopsis genome is transcribed (Martienssen, 2000). These, and other
characteristics (in particular the simplicity of whole plant transformation mediated by
the Agrobacterium tumefaciens T-DNA plasmid) (Katavic et al., 1994; Clough and
Bent 1998) contributed to the selection of Arabidopsis as a designated model
flowering plant by the Security Council of Genetic Organisms in 1998 (Fink, 1998).
Recent work indicating the potential genetic colinearity between A. thaliana and
commercial crucifers validates this designation (Gale and Devos, 1998). The current
large-scale commitment to completely sequencing the Arabidopsis genome is
expected to be fulfilled by the end of the year 2001 (Bevan, 1997; Meinke et al.,
1998). The generation and study of Arabidopsis mutant collections are possible
through chemical mutagenesis of seeds, and also by insertional mutagenesis with
40
T-DNA inserts from A. tumefaciens (Koncz et al., 1992; Koncz and Schell, 1992;
Feldmann et al., 1994; Feldmann, 1998; Galbiati et al., 2000). Targeted mutation
may now also be possible (Altmann et al., 1992; Beetham et al., 1999; Zhu et al.,
1999).
This combination of useful characteristics, global popularity, and database
resources renders Arabidopsis a powerful and accessible tool for plant genetics and
molecular biology, and so an appropriate target organism for this study.
1.7 Concluding comments
There can be little doubt that the primary plant response to UV radiation is
increased shielding, via pigment and wax production, or associated cuticular
thickening. Photoreactivation of UV-induced CPDs and (6-4) photoproducts is
probably the next line of defense up to the point where damage levels exceed the
capacity of photolyases for repair. NER systems likely play a more significant role
when photoreactivation cannot cope. Plant genes that seemingly function in both
NER and transcription have been described. In addition, several putative NER
defective mutants have been isolated, suggesting the existence of a comprehensive
NER system in plants. Table 1.3 presents a summation of the genes or cDNAs
isolated from plants and thought to be involved in an aspect of DNA damage repair,
as at the time of preparation of this document.
Little is known of plant DNA polymerases. By inference from the
characteristics of replicative polymerases from other systems, replication errors and
other lesions would occur which require mismatch correction systems for repair.
Indeed, the isolation of MUTS, MUTL, and other homologues from Arabidopsis
strongly implies the conservation of other mismatch correction genes, and a
homologous system for repair of mismatches in plants. Unrepaired NER and BER
41
substrates probably create a requirement for a translesion synthesis system also
similar to those found in other kingdoms.
The development and characterisation of mutant plant collections has, and
will continue to facilitate gene discovery, and allow complementation studies to
phenotypically confirm putative repair genes isolated through other approaches. The
value of these collections will be increased as mutant characterisation continues, and
if standardisation of nomenclature is embraced.
Plants undoubtedly have mechanisms to cope with current exposures to both
environmental and intrinsic genotoxic agents and insights into these mechanisms are
slowly becoming available. Plant DNA repair pathways will probably be even more
important in the future if climatic predictions regarding ozone depletion are fulfilled.
Thus, an improved understanding of these pathways is important theoretically, and
may also eventually impact on global agricultural and horticultural industries.
1.8 Focus and scope of this study
This investigation evolved during a period of huge change in molecular
biology, and plant molecular biology specifically. This change was due largely to the
global genome sequencing and collation efforts resulting in the genome of
Arabidopsis thaliana being almost entirely sequenced during the writing of this
thesis. The positive impact of these data is immense, and allows genome screening
based on degenerate PCR, as described early in this thesis, to be achieved
electronically, with massive savings of time, reagents and effort generally. Ideally,
identification of a putative homologue and its predicted cDNA allows amplification
of the target sequence by homologous PCR and subsequent cloning and
characterisation of the gene and gene products.
42
The effect of the peculiarities of the generalised plant life cycle on DNA
damage repair and tolerance mechanisms is also unknown. Does the fixed location of
a plant cell, and the consequent inability of metastasis render DNA repair processes
less important in plants than in the case of, for example, a mammal? Likewise, how
does polyploidy affect the requirement for DNA repair? If these factors relieve
pressure on repair systems in the adult plant, does the alternation of generations in
sexual reproduction, involving a haploid phase, impose a requirement for timely
genome repair?
At the time of initiation of this project, data were available from bacteria,
yeast, humans and other mammals regarding the process and function of DNA
damage repair and NER specifically, but aside from circumstantial evidence that it
existed in plants, no hard evidence was available. Proving the existence of
homologous genes could support the hypothesis that DNA damage in plants can be
repaired by a process similar to NER in other kingdoms. Subsequent examination of
the activity of the proteins coded for by these genes in a characterised yeast system
would extend the body of comparative DNA NER knowledge. Further to this, no
information was known about the molecular tolerance of unrepaired nuclear DNA
lesions in plants in general, and Arabidopsis in particular.
Consequently, this investigation aimed firstly to initiate characterisation of
components of a nucleotide excision repair system by which Arabidopsis could
maintain the integrity of its genome in the face of a model environmental stress
(UV-radiation). The Arabidopsis target genes chosen were the homologues of the S.
cerevisiae RAD1 and RAD3 genes. The yeast rad1 mutant typically displays a
mutator phenotype with a spontaneous mutation rate about 7 fold higher than that of
the corresponding isogenic wild-type (Kunz et al., 1990). Specifically, the rad1
43
mutation resulted in increased frequencies of single-base-pair substitution,
single-base-pair deletion, and insertion of the retrotransposon Ty element. Consistent
with loss of NER, it is also UV-sensitive compared to wild-type. RAD1
(XPF/ERCC4 in humans) has a central role in NER as a component of the
RAD1/RAD10 endonuclease complex (Bardwell et al., 1992; Tomkinson et al.,
1993), but is probably not involved in the TFIIH transcription factor (Bardwell et al.,
1994; van Vuuren et al., 1995), and so is not an essential gene. In contrast, RAD3
does function in the basal transcription factor (BTF2/TFIIH) transcription initiation
complex, and also participates in nucleotide excision repair (NER) (Feaver et al.,
1993). Elements of these dual functions can be separated structurally in the protein,
as discussed below in section 3.3.1. Additional functions in recombination and
mismatch repair are indicated by mutational spectra, and other, studies (Montelone et
al., 1988; Song et al., 1990; Yang et al., 1996; Maines et al., 1998). As expected,
yeast strains in which RAD3 has been entirely deleted are non-viable (Naumovski
and Friedberg, 1983).
The Arabidopsis sequences were to be evaluated as homologues on the basis
of nucleotide and protein sequences and domain analysis, and also by their ability to
complement the defects in UV repair and other functions of relevant yeast mutants.
Investigations of other repair genes (RAD2, RAD10) have also been undertaken, to
add to the weight of evidence pointing to conservation of not only individual repair
proteins, but of the overall NER system.
The second aim of this investigation required identification and
characterisation of Arabidopsis homologues of the yeast translesion synthesis
systems. Consequently, screening of the Arabidopsis genome, both in reality by
PCR, and virtually by computerised database searches, was undertaken for
44
counterparts of the Saccharomyces cerevisiae REV3 and RAD30 genes that could
subsequently be cloned and characterised.
These aims have largely been achieved, in that full length RAD1 (atXPF) and
RAD3 (atXPD) homologues from Arabidopsis have been identified, cloned from
cDNA and assayed for the ability to complement a yeast rad1 or rad3 strain,
respectively. Recent publications from independent laboratories largely confirm and
complement the atXPF results described later in this report (sections 3.7 to 3.10). A
putative full length RAD10 (atERCC1) sequence has been identified in the
Arabidopsis database, and the predicted protein sequence evaluated for its similarity
to other RAD10 homologues. A putative RAD2 homologue (atXPG) has also been
identified from the Arabidopsis sequence database, and a fragment of the
corresponding cDNA amplified and sequenced. Again, the predicted protein
sequence has been assessed for comparative sequence conservation. Although none
of these plant sequences derived in this study has thus far been able to complement
the UV-sensitivity of the relevant yeast mutant, the substantial evidence from
sequence analyses in this study, and also from other laboratories during the last four
years, points very strongly to the existence of an NER system in Arabidopsis.
In addition, putative homologues of the yeast TLS genes REV3 and RAD30
have been identified from plant database entries. Again, strong sequence
conservation, as indicated by alignments of the predicted protein sequences, provide
initial evidence for the existence in Arabidopsis of TLS systems comparable to those
found in yeast, mammals, and other organisms. These genes, and atXPG and
atERCC1, and their corresponding cDNA species are parts of the ongoing work
arising from this study.
45
2 Materials and Methods
2.1 Arabidopsis thaliana ecotypes and seed lines
All DNA and RNA preparations in this study were from the Columbia
(Col-o) ecotype, grown from seed stocks held and maintained by our laboratory, and
originally obtained from the Arabidopsis Biological Resource Centre (ABRC) at
Ohio State University, Columbus, Ohio, USA.
2.2 Arabidopsis thaliana growth conditions
Arabidopsis thaliana was grown under controlled conditions on either
commercial potting mix, or on At-medium in 90 mm dispoable Petri dishes. From the
time of sowing, plants were maintained at 21-23oC and 50-80% relative humidity
with a daily photoperiod of 16 h. Conditions were controlled by use of either a
Conviron growth chamber (Model E7, Controlled Environments Limited, Canada),
or a comparable climate-controlled room of our own construction.
2.3 Media
The following formulations apply to liquid broth media (per liter). For plate
media, the formulations were the same with the addition of Difco bacto agar to 20 g/l
(unless otherwise specified). Unless otherwise specified, all media components were
purchased from Life Technologies.
2.3.1 At -medium (derived from Hoagland and Arnon, 1950)
5 ml 1 M KNO3
2.5 ml 1 M KH2PO4
2.0 ml 1 M MgSO4
2.0 ml 1 M Ca(NO3)2
2.5 ml 0.02 M Fe-EDTA
46
1 ml Micronutrient Mix:
70 mM H3BO4
14 mM MnCl2•4H2O
0.5 mM CuSO4
1 mM ZnSO4•7H2O
0.2 mM NaMoO4•2H2O
10 mM NaCl
0.01 mM CoCl2
The pH of the medium was adjusted using NaOH. For solid medium, 7 g of
agar per liter was added.
2.3.2 Luria-Bertani (LB) broth (Sambrook et al., 1989)
10 g Bacto tryptone
10 g NaCl
5 g Yeast extract
2.3.3 YT broth (Miller, 1972)
5 g NaCl
5 g Yeast extract
8 g Bacto tryptone
For ampicillin resistance selective medium (YT+Amp), ampicillin was added
(after autoclaving and cooling of the medium) to a final concentration of
100 µg/ml. For blue/white screening, 160 µg of 5-bromo-4-chloro-3-indolyl-
β-D-galactoside (X-Gal), dissolved in dimethylformamide, was spread on
each prepoured 90 mm YT+Amp plate.
2.3.4 2xYT broth
10 g NaCl
10 g Yeast extract
16 g Bacto tryptone
47
2.3.5 YPD broth (Sherman et al., 1983)
10 g Bacto yeast extract
20 g Bacto peptone
20 g Dextrose
2.3.6 SD (synthetic dextrose) (Sherman et al., 1983)
1.7 g Yeast nitrogen base (without amino acids or ammonium salt)
5 g Ammonium sulfate
40 g Dextrose
Amino acids and nucleic acid bases were added or omitted as required to create
specific selective media to a final concentration of:
L-lysine HCl: 30 mg/litre
L-histidine HCl: 20 mg/litre
L-leucine: 30 mg/litre
L-tryptophan: 20 mg/litre
Adenine sulfate: 10 mg/litre
Uracil: 20 mg/litre
2.3.7 Raffinose expression medium
The formulation of raffinose/galactose expression medium was similar to that
for synthetic dextrose medium, with the replacement of dextrose by raffinose (added
as a filter-sterilised 10% solution after autoclaving and cooling of the medium to
55oC), and addition of galactose (after autoclaving and cooling of the medium to
55oC) to a final concentration of 0.5%.
2.3.8 SOC broth (Sambrook et al., 1989)
2% Bacto tryptone
0.5% Yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2-6H2O
20 mM Dextrose
48
2.3.9 SD-Leu-Trp+FOA selection plates (Boeke et al., 1984)
Add: 0.68 g Yeast nitrogen base
2 g Ammonium sulfate
16 g Dextrose
8 g Difco Agar
L-lysine HCl: to 60 mg/litre
L-histidine HCl: to 40 mg/litre
Adenine sulfate: to 20 mg/litre
Uracil: to 40 mg/litre
H2O to total volume 200 ml and autoclave.
In a separate flask, 0.4 g fluoro-orotic acid was dissolved in 200 ml H2O, and
agitated at 65oC to dissolve, followed by filter sterilisation. Both flasks were
stabilised at approximately 60oC, before combining with thorough mixing, and
pouring.
2.4 Yeast and Bacterial Strains
MKP-o (MATα, can1-100, ade2-1, lys2-1, ura3-52, leu2-3,112, his3-∆200, trp1-∆901) (Pierce et al., 1987)
KAM1 (MATα, can1-100, ade2-1, lys2-1, ura3-52, leu2-3,112, his3-∆200,
trp1-∆901, RAD1::LEU2) (Kunz et al., 1990) WS3-1 (MATα, can1-100, ade2-1, lys2-1, ura3-52, leu2-3,112, his3-∆200,
trp1-∆901, RAD3::rad3-1/TRP1) (Yang et al., 1996) D5225∆∆∆∆’32-38A (MATa, can1-100, his3-∆1, leu2-3, trp1-289, ura3-52,
rad3::LEU2) (Dr. R. Schiestl) INVααααF’ [F’, endA1, recA1, hsdR17, (rk
-,mk+) supE44, thi-1, gyrA96, relA1,
φ80lacZ∆15, ∆(lacZYA-argF)U169 λ-] (Invitrogen) TOP10F’ [F’, {lacIq Tn10 (TetR)} mcrA ∆(mrr-hsdRMS-mcrBC),
φ80lacZ∆M15, ∆lacX74, recA1, araD139, ∆(ara-leu)7697, galU, galK, rpsL (StrR), endA1, nupG] (Invitrogen)
49
2.5 Plasmids
pNF1000 RAD1, 2µm, URA3 (W. Siede)
PCR2.1 lacZα, F1 ori, Kanr, Ampr, (Invitrogen)
pYES2.1HIS-Topo-V5 F1 ori, Ampr, 2µm, URA3, PGAL1 (Invitrogen)
The pYES2.1HIS-Topo-V5 expression system allows direct topoisomerase-mediated
TA cloning of a Taq-generated PCR product into the vector, and control of
expression of the insert from a transformed yeast strain by the presence or absence of
galactose in the growth medium, via the activity of the yeast GAL1 promoter. Since
directional cloning in this system is not possible, recombinants must be screened to
determine the orientation of the insert. Depending on the presence or absence of an in
frame STOP codon in the cloned insert, the protein expressed may be either as the
native sequence, or as a fusion protein incorporating a C-terminal amino acid tag
coded by the vector. This tag includes a poly-histidine motif facilitating purification
of the fusion protein, and also the V5 epitope sequence from the paramxovirus SV5
(Southern et al., 1991), for which a labelled antibody is commercially available.
pSP32 RAD3, 2µm URA3 (R. Schiestl)
pRS95 rad3ts14, ARS1, CEN4, TRP1 (R. Schiestl)
2.6 Isolation of genomic DNA from Arabidopsis thaliana
Genomic Arabidopsis DNA was prepared from leaf tissue by either of 2
standard methods, with minor modifications. Initial maceration of 1 g of fresh plant
tissue was achieved in 3 ml (Method 1) or 1 ml (Method 2) of extraction buffer (EB)
(as described below) using a tissue disrupter of our own design and construction.
50
2.6.1 Method 1
Following the protocol of Chee et al. (1991) the formulation of EB was: 0.1M
Tris-HCL pH 7.5, 0.7 M NaCL, 2 ml 100 mM EDTA, 1% w/v
hexadecylcetyltrimethylammonium bromide (CTAB, Sigma Biochemicals), and 1%
v/v β-mercaptoethanol (BDH Chemicals). The tissue macerate was held at 65oC for
90 min, with occasional mixing. After cooling to RT, 1.5 ml chloroform was added,
the contents of the tube were mixed by inversion, and then centrifuged at 4,500 xg
for 15 min at RT. The top aqueous layer was transferred to a new tube and
re-extracted with 1.5 ml chloroform. The top aqueous layer was again transferred to a
new tube, and an equal volume of 2-propanol was added with gentle mixing. This
preparation was then set aside for > 1 h. Centrifugation at 4,500 xg for > 30 min at
RT followed by washing in 70% ethanol yielded clean DNA suitable for dissolution
in TE containing RNAse A (10 µg/ml, Life Technologies) and subsequent PCR
amplification. Storage in the interim was at –20oC.
2.6.2 Method 2
The second DNA isolation procedure employed was also an adaptation of a
published method (Dellporta et al., 1983). In this case, fresh plant tissue was
macerated in 100 mM TRIS-HCl pH 8.0, 50 mM EDTA, 0.5 M NaCl, 10 mM
β-mercaptoethanol and 1.5% sodium dodecyl sulfate. The suspension was incubated
at 65oC for 30 min with occasional mixing before the addition of 320 µl 5 M
potassium acetate, and incubation at 4oC for about 30 min. Following centrifugation
at 11,000 xg at 4oC for 30 min, the clear supernatant was transferred to a new tube
containing an equal volume of ice-cold 2-propanol. The mixture was then incubated
at 4oC for > 1 h, and again centrifuged at 11,000 xg and 4oC for > 30 min. The
supernatant was decanted, and the nucleic acid pellet washed in 70% ethanol before
51
dissolution in TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.4) containing RNAse
A (10 µg/ml, Life Technologies). This solution was incubated at RT for 30 min, then
extracted twice with TE buffer-saturated phenol. The resulting aqueous fraction was
transferred to a new tube to which was added 0.1 volume of 3 M NaCH3COO and an
equal volume of 2-propanol. The mixture was centrifuged at 11,000 xg and 4oC for
30 min and the resulting pellet was washed with 500 µl of 70% ethanol at RT,
redissolved in TE, and stored frozen at –20oC.
2.7 DNA restriction digests
Restriction enzymes were purchased from Life Technologies, and were
supplied with a matching 10X reaction buffer. In general, reactions were incubated at
37oC for 1 h in 50 µl of 1X buffer with 1-2 µg plasmid DNA and 10-50 U of
enzyme.
2.8 Internet-based utilities
Access to remote molecular biology utilities was via internet links and a
desktop personal computer. Such utilities include:
ENTREZ database http://www.ncbi.nlm.nih.gov:80/entrez
BLAST http://www.arabidopsis.org/cgi-bin/blast/TAIRblast.pl
TBLASTX http:// www.arabidopsis.org/cgi-bin/blast/TAIRblast.pl
TBLASTN http:// www.arabidopsis.org/cgi-bin/blast/TAIRblast.pl
NetPlantGene http://www.cbs.dtu.dk/NetPlantGene.html
Countcodon http://www.kazusa.or.jp/codon/cgi
52
2.9 Sequence comparisons
All protein and nucleotide alignments were achieved using the CLUSTALW
package (Thompson et al., 1994) with default parameters. Output from this package
was maintained in CLUSTAL format, in which a * is used to denote identity at that
position, and : or ⋅ denote conservative replacement. Phylogenetic dendograms were
also generated by CLUSTALW and graphically displayed by TREEVIEW 1.4
(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
2.10 Polymerase chain reaction primers
All PCR and sequencing primers were commercially synthesised at a
40 ηmole scale by either Life Technologies (http://www.lifetech.com) or Geneworks
(http://www.bresatec.com.au), and delivered lyophilised. Primer sequences are
tabulated in Table 2.1.
2.11 Amplification by PCR
PCR amplifications were carried out employing native Taq DNA polymerase
(Life Technologies). The general formulation of the reaction mix, (in 100 µl total
volume), was: 20 mM Tris-HCl pH 8.4, 50 µM KCl, 1.5 µM MgCl2, 0.8 mM each of
dATP, dCTP, dGTP, dTTP, 25 pmol of each primer, and approximately 500 ηg of
genomic DNA template. All reagents were from Life Technologies, except the
amplification primers (section 2.8). PCR programs were carried out using a PTC-100
thermal cycler (MJ Research). A typical program consisted of an initial denaturation
step of 2 min at 93oC, followed by a further 30 cycles; each cycle composed of 30 s
at 93oC, 15 s at the annealing temperature, and 1 min per kb of amplification product
at 72oC. Samples were then held at 6oC until they were removed from the cycler.
53
Cycling conditions including magnesium concentration, temperature profile and
extension time were optimised for each set of primers and template.
2.12 Precipitation of PCR products
When required, DNA generated as a result of PCR amplification was
precipitated by the addition of 0.5 volume of 7.5 M ammonium acetate and 2.5
volumes of RT ethanol (Crouse and Amorese, 1987). After mixing, the solution was
incubated for > 1 h. Centrifugation was for at least 30 min at 11,000 xg and RT.
After aspiration of the supernatant, the pellet was washed with 70% ethanol at RT
and dried under vacuum.
2.13 Isolation of RNA from Arabidopsis thaliana and synthesis of cDNA
Total RNA was isolated from 80 mg of Arabidopsis col-o leaf tissue using the
Qiagen RNeasy Kit, according to the manufacturers instructions, with the exception
that the tissue was macerated in Buffer RLT at room temperature by a mechanical
tissue disrupter of our own construction. In the final step of the procedure, RNA was
eluted by two washes of 30 µl each of warm RNAse-free water. RNA formaldehyde
gel eletrophoresis was as described by Gerard and Miller (1986), with the minor
modification that ethidium bromide was included in the RNA loading buffer at a
concentration of 50 µg/ml. The prepared total RNA (1-5 µg as estimated from gel
electrophoresis) was used as template for either Superscipt II Reverse Transcriptase
(Life Technologies) or Expand Reverse Transcriptase (Roche Molecular
Diagnostics) to synthesise oligo-dT-primed cDNA according to the enzyme
manufacturers instructions.
When purified mRNA was required, it was prepared from 50 µg of total RNA
via the BioRad PolyATract system. Again, elution was by two aliquots (125 µl) of
54
warm RNAse-free water. A 50 µl aliquot, (equivalent to approximately 10 µg of total
RNA), of this mRNA preparation was then used to generate cDNA, again employing
an oligo-dT primer and Superscript II Reverse Transcriptase in a total reaction
volume of 20 µl.
2.14 Preparation of RNA and cDNA from yeast (Saccharomyces cerevisiae)
Total RNA was prepared from yeast cultures in a similar fashion to the
method employed for plant RNA isolation, and utilised components of the Qiagen
RNEasy kit. Briefly, 40 ml yeast culture selective for plasmid retention (uracil-
ommission medium with 2% raffinose as the sugar source and 0.5% galactose) was
spun down at 5,000 xg in a Beckman JA-MI centrifuge fitted with a JA21 rotor.
Supernatant was removed by aspiration, and the cell pellet resuspended in 450 µl of
Buffer RLT (with β-mercaptoethanol) from the Qiagen RNEasy kit. This suspension
was then transferred to a 1.5 ml flip-top disposable plastic tube, and 200 mg of
acid-washed glass beads added. The tube and contents were mixed vigorously for 3
min, before transfer of the contents to the Qiagen Qiashredder column. The process
was then continued as per the Qiagen instructions. RNA was finally eluted from the
RNEasy column by the addition of two aliquots of 30 µl of sterile distilled water
(sdH2O). RNA formaldehyde gel eletrophoresis and cDNA synthesis was as
described in section 2.10 above.
2.15 DNA sequencing
All DNA sequencing (except where specified) was carried out on a
commercial basis at the DNA Sequence and Synthesis Facility, Westmead Institutes
of Health Research, Sydney, on an ABI 370 DNA sequencer, using BIG DYE �
chemistry.
55
2.16 atXPD 3’ RACE
Plant RNA and cDNA were prepared as previously described (section 2.10
above). The cDNA template was then used to PCR amplify DNA bounded by the
primer AtmRNA-Rad3-SF5 on one strand, and the anchor primer AP1 on the other
strand, as depicted in Figure 2.1. All primers used are listed in Table 2.1. The
product of this amplification was diluted 100X and used as template for a nested
PCR using the primers AtmRNA-Rad3-SF7 and Ent100-6. Optimisation of this
nested PCR resulted in the production of a clonable fragment, as judged from gel
electrophoresis results. An aliquot (80 µl) of this PCR product was precipitated
overnight by standard methods with 0.33 volume of 7 M ammonium acetate and 2
volumes of AR grade 99.5% ethanol (Crouse and Amorese, 1987). This precipitate
was collected by centrifugation, washed with 70% ethanol, dried, and TA cloned
(Original TA Cloning Kit, Invitrogen) in an overnight ligation at 14oC into the
plasmid vector pCR2.1 as per the manufacturers instructions. Transformation into
bacterial strain InvαF’ allowed clonal selection and further processing, including
plasmid isolation, diagnostic PCR, and sequencing.
2.17 Yeast Transformation
Yeast transformation was achieved via a procedure based on a combination of
published methods (Ito et al., 1983; Gietz et al., 1992; Agatep et al., 1998). Briefly, 1
ml of an overnight YPD yeast culture was centrifuged at RT for 2 min at 2,400 xg to
yield a cell pellet, which was washed by vigorous resuspension in 100 µl of sdH2O.
The cells were again pelleted at RT and 2,400 xg for 2 min in a Heraeus benchtop
centrifuge, and the supernatant removed. To the cell pellet was added (in this order):
240 µl polyethylene glycol 3350 (50% w/v), 36 µl lithium acetate (1 M), 50 µl yeast
56
tRNA (5 mg/ml), and from 1 to 5 µg plasmid DNA in 34 µl sdH2O to a total volume
of 360 µl. Cells were resuspended by vigorous mixing and then incubated at 42oC for
20 min. This suspension was then centrifuged at RT and 11,000 xg for 5 min, and the
supernatant discarded. Cells were resuspended in 200 µl sdH2O, and 100 µl applied
to each of two SD-Ura plates (unless otherwise specified). After 3-5 days incubation
at 30oC, individual colonies were harvested, spot-tested, and frozen at –80oC in 50%
glycerol.
2.18 UV-Sensitivity complementation studies in yeast
Yeast strains carrying recombinant expression plasmids were grown (see
below) in selective broth medium (S-Raff-Ura+0.5% Gal) under conditions to
express the plasmid insert. Such conditions were validated by the complementation
performance observed in the yeast mutant strain when the expression vector carried
the yeast gene as a positive control insert. This gene was cloned such that it was
orientated correctly behind the Gal1 promoter. The plant cDNA to be tested was
similarly oriented in the expression vector. A third insert was also cloned into the
expression vector and subjected to the same complementation assay, this insert
consisted of a plant cDNA in its reverse orientation (negative control). Cultures were
grown at 30oC with shaking to a titre of about 5 x 106 cells/ml. Each culture was
counted by Coulter Counter (Model Z1) and then suitably diluted in sdH2O for
plating onto solid expression medium and UV-exposure. UV-C exposure under dim
room lighting was to a 60 cm UV tube (Davis Ultraviolet Cat No. G36T6L) mounted
in a casing of our own construction. Dose was measured by a UVX Radiometer
(Ultraviolet Products) meter with a UVX-25 probe. UV-C irradiance at the surface of
the medium was reduced to approximately 0.40 J/m2/s by inserting layers of mesh
between the plate and the source. Once irradiated, plated cultures were immediately
57
wrapped in foil (under dim light to minimise photoreactivation) and incubated at
30oC for 5 days before scoring.
59
3 Results
3.1 cDNA library screening for an A. thaliana XPD homologue
A protein alignment as shown in Figure 3.1 allowed identification of
conserved regions of the RAD3/XPD proteins against which to design degenerate
PCR primers RAD3-F1 (amino acid target sequence: EMPSGTG) and RAD3-R1
(amino acid target sequence: DEAHNID) as shown in Table 2.1. These degenerate
primers were then used to amplify a fragment approximately 850 bp in length from
about 500 ηg of genomic Arabidopsis DNA. Automated sequencing of this
fragment revealed a nucleotide sequence suggestive of a RAD3 homologue, the first
indication that the Arabidopsis genome included such a homologue (subsequent
comparison with the atXPD gene retrieved in this study showed this fragment to
correlate with the region of the atXPD gene bound by base pairs numbered 121 and
846).
The nucleotide sequence of this genomic fragment, in conjunction with
protein alignments to identify conserved, translated sequence, allowed screening of
a commercial Arabidopsis thaliana cDNA library library (Stratagene Cat. No.
937010) for the presence of an atXPD homologue. This was undertaken using the
“GeneTrapper” system (Life Technologies), with modifications as described by
Shepard and Rae (1997). Capture (RAD3-Cap) and blocking oligonucleotides
(RAD3-5’Block and RAD3-3’Block) are shown in Table 2.1.
The plasmid insert sequence retrieved by 3 rounds of screening showed
large areas of identity with an Arabidopsis database clone when used as a query
sequence in a BLASTN search (see section 2.8) (Altschul et al., 1997). The
Accession number of this Arabidopsis clone was AB026661, listed as a BAC
60
genomic sequence of unknown function from chromosome 5. Unfortunately, neither
the RAD3-Capture sequence, nor the blocking oligonucleotide sequences were
included in the sequence of the retrieved clone. Also the predicted protein
sequences resulting from translation in all 6 reading frames of this clone bore no
resemblance to other RAD3 homologues. The fact that a single clone appeared to
constitute the majority, if not all, of the enriched plasmid pool indicated that the
GeneTrapper system was imposing some selectivity on the constituency of the
resulting plasmid pool. However, the absence of the target sequence in the retrieved
clones clearly indicated that the stringency of the system was insufficient.
Alternatively, it may have been that the problem with the approach was not a matter
of stringency, but simply that the target clone was not represented in the library at a
retrievable level. A below-specification library titre (approximately 1 order of
magnitude lower than the manufacturers specification was found in this case) may
also have contributed to this. A second, independent library screening attempt,
performed on a commercial basis by Genome Systems Inc. (St. Louis, Mo. USA)
also failed to retrieve a RAD3 cDNA homologue from the Arabidopsis library
favoured by that company. Consequently, it was decided that library screening was
not the most appropriate approach, and so the focus of this investigation was
changed to an attempt to retrieve a RAD3 homologue from A. thaliana cDNA
directly from RNA preparations rather than from a cDNA library.
3.2 Virtual screening for an A. thaliana XPD homologue
The nucleotide sequence of the genomic fragment described in section 3.1
was used as a virtual probe to electronically screen the existing Arabidopsis
thaliana DNA sequence database at Stanford University via the TBLASTX
algorithm (see section 2.8). This revealed extensive sequence homology with a
61
portion of chromosome I genomic clone AC005278, from base number 56,552 to
56,916. Retrieval of the sequence of this genomic clone and visual comparison of
the 3 translations of the plus strand (on the basis of the BLAST result) with other
RAD3 homologues resulted in selection of a 7 kbp portion of this sequence
(including the retrieved fragment plus surrounding sequence) for further analysis.
No duplicate copies of this fragment at any other genomic locations have been
observed in the Arabidopsis database to this time. This sequence was submitted to
NetPlantGene (see section 2.8) in an attempt to precisely predict intronic and exonic
regions, thereby allowing the design of PCR primers to specifically amplify the
Arabidopsis XPD homologue from cDNA. Following optimisation of the PCR
process, the amplified product was T/A cloned into pCR2.1 and each strand was
sequenced. Sequencing primers are listed in Table 2.1, the relative sequencing
direction is identified by the “SF” or “SR” suffix.
3.3 Characterisation of the atXPD genomic, cDNA and amino acid sequences
The genomic atXPD sequence selected from clone AC005278 is shown in
Figure 3.2. The corresponding cDNA sequence was determined, and is also shown
in Figure 3.2, along with the predicted protein sequence.
The atXPD cDNA is 2,277 nucleotides in length. Comparison with the
atXPD genomic sequence revealed that the gene is comprised of 11 introns and 12
exons. “Highly confident” predictions by NetPlantGene (see section 2.8) based on
the genomic sequence (AC005278) correctly specified 13 intron/exon junctions,
another 6 junctions were not predicted at this confidence level. Two junctions were
not predicted at all, these being at genomic nucleotides 1,336/7 and 3,183/4 (exons
3 and 9). Table 3.1 describes the introns and exons of the atXPD gene sequence.
Average exon size is 190 bp. The average intron size is 160.5 bp, somewhat shorter
62
than the Arabidopsis genome-wide figure of 240 bp (Deutsch and Long, 1999). This
gene has 563.7 bases of exonic sequence, and 436.3 bases of intronic sequence per
kilobase of genomic sequence.
The atXPD cDNA is 44% GC and codes for a protein of 759 amino acids.
An alignment of the predicted protein sequence with other RAD3/XPD homologues
is displayed in Figure 3.3. In the multiple alignment shown, atXPD exhibits
consensus conservation of amino acids across the alignment at 35% of positions,
this figure increases to 69% if conservative replacements are included. Compared to
the human XPD sequence only, atXPD is very highly conserved, with identical
residues at 410 of 759 positions (54%), and conservative replacement at a further
216 positions (28%), totalling to 82% conservation overall. Seven conserved
domains have been identified in RAD3 and RAD3 homologues as being
characteristic of helicases (Deschavanne and Harosh, 1993), in keeping with a
suggested helicase function. These regions also are shown in Figure 3.3, in coloured
italics. The Arabidopsis protein atXPD shows 100% identity or conservative
replacement in domains I and IV, and recognisable conservation in other domains,
lending credibility to the notion of homologous function.
3.3.1 atXPD 3’-RACE amplification and sequencing
The nucleotide sequence generated from the atXPD 3’-RACE procedure is
displayed in Figure 3.2. This process provided confirmation that the STOP codon
thought to be active on the basis of intron/exon predictions and protein alignments
was in fact in frame and probably in use. This mRNA includes 153 bases of
untranslated sequence between the STOP codon and the poly-A tail. The first 12 of
these bases immediately downstream of the STOP codon conform to the
characteristic A/T richness commonly found in this region (Joshi, 1987). The
63
ubiquitous eukaryotic AATAAA polyadenylation signal (Joshi, 1987) could not be
located in the 3’ untranslated region of atXPD. However, the higher plant consensus
sequence of tATTG (Joshi, 1987), normally located immediately upstream of the
AATAAA motif and thought to have some function in pre-mRNA processing is
perhaps represented in the atXPD sequence as AATTG (44 bases upstream of the
poly-A tail in the mature transcript). The A/T substitution at the first position of this
consensus sequence occurs slightly less than 25% of the time (Joshi, 1987). If
AATTG in atXPD is homologous to tATTG, then this suggests that the sequence
ATTTAA, slightly downstream, is the atXPD equivalent of the AATAAA
polyadenylation signal. Two base mismatches such as observed in this sequence
have previously been seen in only 3 of 46 genes (7%) (Joshi, 1987). Potential
explanations for this variation include the possibility that the previous compilations
have been based on a small number of genes (46 sequences), and so may not be
representative. Alternatively, it has been suggested that the AATAAA sequence
may not be absolutely required for polyadenylation of plant mRNAs (Mogen et al.,
1990). If so, this may represent a fundamental difference in RNA processing
between plants and animals.
3.4 Translation initiation in atXPD
The atXPD cDNA relies on GAAGAUGAUCUUU as the sequence context
of the translation initiation codon. Comparison with the consensus dicot sequence
A(A/C)aAUGGC (Joshi et al., 1997; Lukaszewicz et al., 2000) reveals several
differences. Although the most often invariable A at position –3 is present, the usual
G at position +4 (67% of dicotyledonous plant sequences) has been replaced by an
A, a substitution seen in 15% of dicot sequences (Joshi et al., 1997). The functional
significance of this replacement in vertebrates has been suggested to extend to
64
control of the levels of proteins potentially detrimental to the cell (Kozak, 1991).
This notion has been very tentatively extended in a survey of higher plant
sequences, where, of 114 sequences having an A at position +4, 43 were found to
code for transcription factors/signal transducers or regulatory proteins (Joshi et al.,
1997).
The observed substitution of U for C at position +5 occurs in only 11% of
known dicot sequences, however the consequence of this and other downstream
substitutions, is not yet known. No putative TATA box (compliant with the
eukaryotic consensus sequence TATA(T/A)A(T/A) (Breathnach and Chambon,
1981)) was identified in the 5’ untranslated region of the atXPD gene, although
possible variants of this sequence exist at genomic nucleotides –406 to –412
(TATACAT), -462 to –468 (TATATTT), and –934 to –940 (TATACAC).
3.5 atXPD complementation assay for UV-sensitivity in yeast
The atXPD cDNA was tested in vivo for its ability to complement a
repair-specific mutation (rad3-1) in the yeast RAD3 gene which confers sensitivity
to UV radiation but does not preclude viability. The complete plant cDNA was
cloned into the pYES2.1HIS-Topo-V5 expression vector as previously described in
section 2.17. Since the cDNA included the native STOP codon, it was not expressed
as a fusion protein so as to minimise the impact of factors other than the presence of
the plant cDNA. The positive control constructed was the yeast RAD3 gene
including the region extending from 3 bases upstream of the START codon to the
last base before the STOP codon. This construct expresses a fusion protein, as this
approach was thought to have potential advantages if this construct failed to
complement. These advantages included proof of transcription in a Northern blot
using an oligonucleotide probe directed to the poly Histidine/V5 tail of the fusion
65
mRNA, and proof of translation by Western blot analysis of protein extracts
employing the commercially available anti-V5 epitope antibody. In fact, these
advantages were not utilised, as this positive control RAD3 construct performed as
expected. A negative control consisting of the atXPD cDNA cloned in the reverse
orientation (fusion protein) was used to evaluate the influence of the expression
system itself.
The transformed yeast rad3-1 mutant strains expressing either the wild-type
yeast RAD3 gene, the plant atXPD cDNA or the negative control insert (atXPD
cDNA in reverse orientation) were assessed for their ability to survive a UV-C
challenge when grown under conditions to induce expression. Results of this
challenge are presented graphically in Figure 3.4. Neither the presence of the
negative control insert nor atXPD in the expression vector added to the
UV-resistance of the mutant strain. In contrast, expression of the wild-type yeast
RAD3 gene restored the UV-resistance of the mutant to wild-type levels, confirming
the efficacy of the expression system. Transcription and translation of the atXPD
insert from the expression vector was demonstrated by the results described in the
following section.
3.6 atXPD complementation assay for viability in yeast
RAD3 and its homologues are essential genes due to their involvement in
transcription, which is a function distinct from their role in repair (see section 4.1).
Since transcriptional competence is required for viability, a temperature-sensitive
yeast rad3 mutant was used for the assay. In this way, the essential function of the
resident RAD3 protein could be turned on or off in this strain by temperature
change. The laboratory of Dr. Robert Schiestl at Harvard University very kindly
provided such a strain (D5225∆’32-38A with plasmids pSP32 and pRS95). This
66
yeast strain has a chromosomal rad3 deletion, created by incorporating a
RAD3::LEU2 deletion cassette into a strain already carrying pSP32. Plasmid pSP32
(Reynolds et al., 1985) carries the wild-type RAD3 gene and confers a URA+
phenotype, and pRS95 (Guzder et al., 1994), a second plasmid transformed into the
strain, carries the rad3ts14 mutant allele (see section 4.1.2) and confers a TRP+
phenotype (see section 2.5 for genotypes). Effectively, this strain is a rad3 null
chromosomal mutant and the mutation is complemented fully by the wild-type
RAD3 allele carried on pSP32. In this situation, the rad3ts14 mutant is not required
for viability, but the presence of the pRS95 plasmid could be maintained by growth
on the appropriate omission medium.
With both pSP32 and pRS95 plasmids present, the strain is viable at either
30oC or 39oC. To assess the ability of the atXPD cDNA to confer viability, the
pSP32 plasmid (and the wild-type RAD3 gene) was replaced by the
pYES2.1HIS-Topo-V5 expression vector carrying atXPD. To achieve this, the
strain carrying both pSP32 and pRS95 plasmids was initially grown in leucine and
tryptophan omission broth (see section 2.3 for descriptions of media) at 30oC to
logarithmic growth phase and then plated on similar solid omission medium with
the addition of fluoro-orotic acid and uracil to select against the URA+ pSP32
plasmid (Boeke et al., 1984). Subsequently, D5225∆’32-38A with pRS95 alone
displayed uracil auxotrophy and the expected temperature-sensitive phenotype (see
below), irrespective of the carbon source in the medium. D5225∆’32-38A with
pRS95 and pSP32 displayed similar viability at both temperatures on all media, as
expected. These two observations confirmed that the strains were viable, and
exhibited behaviour consistent with the presence of each of the two yeast RAD3
alleles.
67
D5225∆’32-38A with only pRS95 was then transformed with the
pYES2.1HIS-Topo-V5 expression vector (URA+) carrying either the yeast RAD3
gene or the atXPD cDNA, and plated on raffinose/galactose-based uracil and
tryptophan omission medium to select for colonies carrying both plasmids.
Resulting colonies were frozen in 50% glycerol and the phenotypic markers
subsequently checked. A compliant colony from each of three strains
(D5225∆'32-38A with: 1. pRS95 alone; 2. pRS95 and pYES2.1 HIS TopoV5
carrying RAD3; and 3. pRS95 and pYES2.1HIS TopoV5 carrying atXPD) was then
used to inoculate tryptophan and uracil omission induction medium, or tryptophan
omission induction medium for the strain with pRS95 only. The cultures were
grown to about 106 cells per ml, and for each of the three strains 2,000 cells were
spotted onto a sector of a plate containing expression medium and also onto sectors
of plates containing control medium. A duplicate of each plate was made and one
set of plates was incubated at 30oC, and the other set at 39oC. All plates were
visually inspected after 5 days incubation.
D5225∆’32-38A with pRS95 and pYES2.1HIS-Topo-V5 carrying RAD3
was viable at both temperatures, indicating that the pYES2.1HIS-Topo-V5/RAD3
construct was producing a functional RAD3 protein in such amounts as to confer
relatively normal levels of viability (Figure 3.5). Surprisingly, however, this result
was seen on both raffinose and dextrose-based plates; the latter being a medium
expected to suppress expression of the insert by the GAL1 promoter on
pYES2.1HIS-Topo-V5. Two possible explanations for this phenomenon are that the
GAL1 promoter is “leaky” and maintains a sufficient level of expression even under
maximum suppression, or alternatively that enough RAD3 protein to confer
viability is carried through from the expression culture in which the cells were
68
originally grown before plating. Either way, restored viability at 39oC indicates that
wild-type RAD3 is available to the transcription complex in this transformed strain.
The results of the atXPD assay were also surprising in that the viability of
D5225∆’32-38A with pRS95 and pYES2.1HIS-Topo-V5 carrying the Arabidopsis
cDNA approximated that of the RAD3-complemented strain at both 30oC and 39oC
(Figure 3.5). At 30oC this result can be explained by the presence of the yeast
pRS95 ts allele. However, at 39oC the substantially increased viability must be
conferred by atXPD. This requires that the atXPD protein be incorporated into the
yeast TFIIH complex, and furthermore that it maintains the critical protein
interactions necessary for transcription. Again, complementation for viability was
seen on both raffinose and dextrose media.
To further investigate growth at 39oC on dextrose-containing medium, the
two strains carrying pYES2.1HIS-Topo-V5 derivatives were inoculated directly into
dextrose-based medium without prior culture in expression medium. Both strains
grew at 39oC (data not shown), suggesting that leakiness of the promoter was
sufficient for viability. The wild-type yeast GAL1 promoter has previously been
shown to be incompletely suppressed on glucose medium (Elledge et al., 1991),
resulting in a basal level of expression of about 1/500 that seen under induction
conditions. The pYES2.1HIS-Topo-V5 vector from which these recombinant
plasmids were constructed carries the wild-type Upstream Activating Sequences
(UASG) required for the normal performance of the GAL1 promoter (West et al.,
1984). Consequently, GAL1 promoter leakiness in this expression vector cannot be
attributed to the absence of this control sequence. Also, it has been demonstrated
that galactose is released by the breakdown of agar to a free concentration of about
0.06% in 2% agar medium during normal medium sterilisation by autoclaving
69
(Kaplan et al., 1997), although glucose repression should dominate any induction by
this galactose. Overall, in conjunction with the multicopy nature of the vector
(conferred by the 2 µm origin) the leakiness of the GAL1 promoter may result in
sufficient transcription of the insert to restore viability.
In order to make a quantitative assessment of the impact of these
recombinant expression plasmids on the viability of the rad3ts14 strain, the growth
rates in broth cultures were determined. Expression medium broth was inoculated to
about 1x105 cells/ml with either D5225∆’32-38A with pRS95, D5225∆’32-38A
with pRS95 and pYES2.1HIS-Topo-V5/RAD3, or D5225∆’32-38A with pRS95 and
pYES2.1HIS-Topo-V5/atXPD. Cultures were incubated with shaking at 30oC for
10 h to a titre above the lower limit of accurate resolution of the Coulter Counter. At
20 h after inoculation, the three cultures were shifted to incubation at 39oC with
shaking, and the culture titres monitored with time. The results of this procedure are
displayed graphically in Figure 3.6, and reveal a distinct difference between the
maximum growth rate and final titre of D5225∆’32-38A with pRS95 (rad3ts14; 8.11
h/generation; 1.2x106 cells/ml), compared to the maximum growth rate and final
titre of D5225∆’32-38A with pRS95 and either pYES2.1HIS-Topo-V5/RAD3
(rad3ts14 RAD3; 5.40 h/generation; 8.1x106 cells/ml) or
pYES2.1HIS-Topo-V5/atXPD (rad3ts14 atXPD; 4.63 h/generation; 7.5x106
cells/ml). Clearly, the expression of either RAD3 or atXPD is having a positive
impact of roughly equal magnitude on the growth of this strain. The maximum cell
density in these cultures is, however, still less than would be expected, as indicated
by the finding that growth of these cultures at 30oC (Figure 3.7) results in maximum
titres of 1.6x107, 2.5x107, and 2.4x107 cells/ml for the rad3ts14, rad3ts14 RAD3, and
70
rad3ts14 atXPD strains, respectively. The implications of these results are discussed
further in section 4.2.
To examine the possibility that the presence of two RAD3 homologues in
combination in each cell (i.e., the temperature-sensitive rad3ts14 and either the
wild-type yeast RAD3 or Arabidopsis atXPD proteins) may be having some
combinational effect, construction of the equivalent strains without the rad3ts14
allele but with the pYES2.1HIS-Topo-V5 vector carrying either the yeast RAD3
gene or the atXPD cDNA was attempted. This was done by adding tryptophan to
the medium, and so rendering the function of the TRP+ auxotrophic marker on the
pRS95 vector redundant. To do this, D5225∆’32-38A with pRS95 and either RAD3
or atXPD in pYES2.1HIS-Topo-V5 were grown in induction medium with
tryptophan at 37oC. Each strain was then plated at a density of 200 cells/plate on
induction medium plates containing tryptophan. Some of the resulting individual
colonies were then assayed for ability to grow on induction medium plates without
tryptophan compared to induction medium plates with tryptophan. Colonies on all
plates were counted, those that were TRP+ were assumed to have retained pRS95,
those that were TRP- were presumed to have lost pRS95. D5225∆’32-38A with
pRS95 and atXPD was also subsequently plated at a similar density on six induction
medium plates. After five days incubation, the resulting colonies from each plate
were replica-plated onto induction medium with or without tryptophan (six plates
each), and incubated for a further two days at 30oC.
Viability of strains lacking pRS95 would require absolutely that the
expression plasmid insert code for a protein able to functionally complement the
chromosomal RAD3 deletion. With D5225∆’32-38A carrying the wild-type RAD3
gene, 6 of 25 (24%) colonies tested were TRP-. For D5225∆’32-38A:atXPD, visual
71
inspection of the replica plates allowed selection of 375 distinct, single colonies.
Each of these colonies was found to have retained a TRP+ phenotype, as indicated
by growth of the corresponding colony on the tryptophan omission medium. The
absence of TRP- colonies suggests the intriguing possibility that both the rad3ts14
allele and the atXPD cDNA may be required for viability, as discussed further in
section 4.2.
3.7 Virtual screening for an A. thaliana XPF homologue
Protein alignments of available RAD1 homologues revealed considerable
conservation of amino acid sequence across human and mouse proteins, and to a
lesser extent with the S. pombe protein. The S. cerevisiae protein is somewhat less
well conserved, but still shows strong, localised homology (Figure 3.8). Previous
experience with atXPD suggested that mammalian sequences may be closer to the
corresponding plant sequence than is the budding yeast sequence. Consequently, the
human XPF protein (Accession Number: 2842712) was used as a probe to search
the A. thaliana database using the TBLASTN utility (see section 2.8). Since the
highest-scoring sequence identified by this process was derived from a genomic
clone (Accession Number AB010072), an attempt was made to predict intron/exon
boundaries by submitting the putative RAD1 homologue gene sequence to the
NetPlantGene World Wide Web server (Hebsgaard et al., 1996). Based on these
predictions, exonic PCR primers were designed and employed to amplify and
sequence an atXPF homologue from cDNA.
3.8 PCR amplification and cloning of an A. thaliana XPF homologue
All primers were directed towards predicted exonic sequences. The reverse
primer AtmRNA-RAD1-R1 encompassed the ATG codon at what was inferred to
72
be the translation start site. The forward primer AtmRNA-RAD1-F1 was directed
toward the coding region for the protein sequence IQDLFQS, which encompasses a
relatively well-conserved region from the protein alignment (see Figure 3.8). On the
basis of the predicted cDNA sequence, these primers were expected to result in a
PCR product of about 2.4 kbp. Nucleotide sequences of all PCR primers, and
subsequent sequencing primers are shown in Table 2.1.
Optimised amplification from A. thaliana cDNA using the primer pair
AtmRNA-RAD1-F1/R1 yielded a discrete product within the size range expected
from the intron/exon predictions, as visualised by agarose gel electrophoresis (data
not shown). Fresh (<24 h post-amplification) PCR products were ligated overnight
at 16oC into the PCR2.1 TA cloning vector (Original TA Cloning Kit, Invitrogen).
Cloned inserts were screened by diagnostic PCR with internal gene specific primers
and restriction mapping, allowing selection of a compliant clone for automated
sequencing. This resulted in a 2,395 bp putative A. thaliana partial cDNA RAD1
homologue (patXPF). patXPF was fully sequenced commercially in both directions
and the results largely confirmed the intron/exon predictions.
The complete cDNA was subsequently Taq-PCR amplified using the primer
pair AtmRNA-RAD1-F7/R1, primers which straddle both the putative TGA stop
codon (derived from the gene sequence and the cDNA sequencing) and the ATG
start codon, respectively. The resulting amplification products were then cloned
directly into the yeast expression vector pYES2.1HIS-Topo-V5 (Invitrogen). Since
directional cloning into this vector was not possible, recombinant plasmids were
screened to determine the orientation of the insert in the vector. This was done by
using PCR with a combination of vector and gene-specific primers amplifying DNA
73
from bacterial plasmid minipreps, and revealed the presence of inserts of various
lengths, as discussed below.
3.9 atXPF exhibits variable transcript processing
• Three separate cDNA species (atXPF-1, 2, 3) were retrieved from a single pool
of A. thaliana total RNA by oligo dT-primed reverse transcription, and PCR
with homologous gene-specific primers. The cloned cDNAs were sequenced in
entirety in both directions. Within the variation discussed below, each cDNA
sequence confirmed the other, and was highly consistent with the genomic
sequence and also the UVH1 sequence subsequently published (Liu et al., 2000).
These three atXPF molecules varied only in the intron/exon processing of the 3’
region of the cDNA (see Figures 3.9 and 3.10), resulting in lengths of 2,776 bp,
2,924 bp and 2,871 bp, respectively. Since the variation in these cDNA species
is totally consistent with variable intron processing of the mRNA, it is thought
that each cDNA genuinely represents a mRNA transcript, i.e., the observed
variation does not reflect errors in the reverse trancription process of creating
the cDNA pool. In addition, this same result was seen on each occasion that the
amplification was performed, indicating that the observed phenomenon was
independent of the specific RNA preparation or cDNA synthesis. Unfortunately,
the small differences in transcript size make discrimination in a Northern blot
unreliable, as was found, for example, in the case of variable processing of
ANP1 transcripts (Nishihama et al., 1997). Interestingly, the uvh1-1 mutation
also potentially involves a variation in the mRNA transcript processing, as
discussed in section 4.3. The cDNA sequence of atXPF-3, the cDNA species
coding for the longest of the three possible atXPF proteins, is presented in
Figure 3.11 with its predicted protein translation.
74
Screening of the A. thaliana database with the atXPF-3 sequence as probe
returns only a single genomic location (on chromosome 5), again consistent with
production of the observed multiple transcripts simply by variable processing in
vivo of the primary transcript.
3.9.1 Structural features of atXPF cDNAs
All three atXPF cDNAs are comprised of 9 exons and 8 introns (see Table
3.2 for tabulation of the sizes of the introns and exons in atXPF-3). Variation occurs
at the 5’ end of exon 6, where atXPF-2 has a 53 bp extension compared to atXPF-1
and atXPF-3 (Figures 3.9 and 3.10). This extension alters the downstream reading
frame in this clone such that a stop codon is encountered a further 22 codons from
the 3’ end of this extension. The clone atXPF-2 thus codes for a truncated 727
amino acid protein (Figure 3.12). The second variable region is at the 3’ end of exon
7, where atXPF-3 and atXPF-2 have a 95 bp extension of exon 7 compared to
atXPF-1. Loss of this extension and the associated downstream frameshift in
atXPF-1 results in a stop codon being encountered three codons into exon 8.
atXPF-1 consequently also codes for a truncated protein, of 783 amino acids
(Figure 3.12). The sections of RNA sequence that are included or excluded by this
variable processing have an A/U content of 66% in intron 5 and 60% in intron 6,
both consistent with typical dicot intronic sequences (Simpson and Filipowicz,
1996), and at this level, giving no indication of unusual sequence features in these
regions that may contribute to this variability.
Protein alignments indicate that clone atXPF-3 codes for a 957 amino acid
protein most similar to XPF/RAD1 homologues from other organisms (Figure 3.13).
However, the possibility that all or either of the three possible atXPF proteins may
be active in a specific function and/or specific tissue cannot be discounted.
75
Comparison of the atXPF-3 protein with the human XPF protein reveals identical
amino acids at 346 of 957 sites (36%), and conservative replacement at a further
336 sites (35%).
The relevant intron/exon boundaries of the two variable regions of atXPF
clones are detailed in Table 3.3. While variation from consensus sequences
naturally occurs, it is interesting to compare sequences I and II with V, and III and
IV with VI in more detail. Junctions I, II, III and IV all comply with consensus
sequences of lower frequency (and perhaps lower efficiency of splicing), which may
contribute to this variability in processing. The presence of the canonical GU-AG
borders at the termini of the introns is consistent with processing of these borders by
a U2-dependent spliceosome (Brown and Simpson, 1998).
The average exon size for this gene is 319 bp with a standard deviation of
282 bp (Table 3.2). This compares with an average figure genome-wide of 68 bp
(SD = 77 bp) for A. thaliana. Average intron size for this gene is 123 bp (SD =
63 bp) compared to 240 bp (SD = 268 bp) genome-wide for A. thaliana (Deutsch
and Long, 1999). About 9% of characterised A. thaliana introns fall into a size
range of 120-150 bp (Brown et al., 1996). Overall, exons are larger for this gene
than the genomic average whilst introns are smaller, resulting in a more compact
gene structure. No putative polyadenylation signal AATAAA (Mogen et al., 1990)
could be found within the atXPF-3 cDNA. However, this absence has been
observed in other plant transcripts, including atXPD, as discussed in section 3.3.1.
3.9.2 Translation initiation in atXPF
From a combination of gene and cDNA data, the sequence surrounding the
translation initiation codon AUG in atXPF mRNA is UUCAAUGGCGCUG,
differing just slightly from the consensus dicotyledon translation initiation context
76
of A(A/C)aAUGGC (Joshi et al., 1997; Lukaszewicz et al., 2000) by having uracil
instead of adenine at the most often invariable –3 position. A pyrimidine at –3 is
considered functionally unfavourable (Joshi et al., 1997), but nonetheless occurs in
higher plants in various contexts at a frequency of 18% or more (Joshi et al., 1997),
and has been assessed for its impact on function. For example, the initiation
sequence AUCAAUGGC occurs at a frequency of 1.37% in a Genbank dicotyledon
database subset, and was found to have a translation initiation activity in tobacco
protoplasts of about two-thirds that of the consensus sequence (containing A at
position -3) (Lukaszewicz et al., 2000). Vertebrate studies have shown an even
greater decrease in translation rate due to substitution of purines by pyrimidines at
positions –3 and +4. This may represent a further level of functional control,
perhaps relating to protein toxicity (Kozak, 1991), tissue specificity or
developmental phase (Simpson and Filipowicz, 1996).
A TATA-like sequence (Breathnach and Chambon, 1981) constituting a
putative transcriptional control box (TATAAAA) was found at nucleotides number
–792 to -786 (relative to the ATG START codon) in the atXPF genomic sequence,
however, no transcription initiation sequence was identified. This long 5'
untranslated sequence is consistent with the findings of Liu et al. (2000), who
determined that the virtually identical UVH1 cDNA had a 5' untranslated leader
sequence of more than 500 nucleotides. They also suggested the presence of a
putative TATA box, located 742 nucleotides upstream of the ATG START codon.
3.10 Confirmation of the nucleotide sequence of yeast RAD1
In the process of constructing a positive control for the UV complementation
assays (S. cerevisiae RAD1 gene carried on the pYES2.1HIS-Topo-V5 expression
vector; see section 3.11), it became apparent that two conflicting sequences of
77
RAD1 were simultaneously present in the Genbank database; YScRAD1 (Reynolds
et al., 1987) and YScRAD1A; (Yang and Friedberg, 1984). The major difference
between these two sequences is in the carboxyl terminal end of the corresponding
proteins. YScRAD1A extends beyond YScRAD1 by 126 amino acids. This
extension is brought about by a stop codon at nucleotide positions 2916-2918 in
YScRAD1 being out of frame in YScRAD1A. Minor differences also exist with
respect to single base-pair substitutions and deletions/insertions.
To ensure that the complete RAD1 coding region was used as the positive
control, it was necessary to sequence this region of the RAD1 gene from
S. cerevisiae. This genomic fragment, extending bidirectionally well beyond the
coding region, had previously been cloned into the vector Yep24 (Carlson and
Botstein, 1982) to create the plasmid subsequently named NF1000 (Yang and
Friedberg, 1984). Preparation of this plasmid from bacterial culture by standard
methods in our laboratory produced a ready substrate for sequencing, using primers
shown in Table 2.1 (pNF1000-SF1 and pNF1000-SR1). The sequence corrections as
specified in Table 3.4 can now be made, confirming the sequence YScRAD1A to be
correct. Consequently, this sequence was employed for construction of positive
controls.
3.11 atXPF complementation assays for UV-sensitivity in yeast
For evaluation of the ability of atXPF-3 to complement a yeast rad1
mutation in vivo, the pYES2.1HIS-Topo-V5 expression vector carrying the atXPF-3
cDNA, cloned in the correct orientation relative to the promoter, was transformed
into KAM1, a rad1 mutant yeast strain. This construct was designed to include the
atXPF STOP codon, and so does not result in an atXPF fusion protein. Details of
the cloning and transformation procedures followed are as described previously in
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section 2.17. In this instance, however, the positive control insert was the yeast
RAD1 open reading frame (from the START codon to the base immediately
preceding the STOP codon), again expressed as a fusion protein as in the case of the
RAD3 positive control (see section 3.4). The negative control, to examine the
influence of the plasmid vector itself, was the A. thaliana XPD cDNA cloned in the
reverse orientation as described in section 3.4, again expressing a fusion protein.
3.11.1 atXPF UV-sensitivity assay results
The UV-sensitivity of the KAM1 rad1 mutant when transformed with the
expression vector carrying the yeast RAD1 coding region approximated that of the
isogenic wild-type NER proficient parental strain (MKP-o). When the vector carried
a negative control insert, UV-sensitivity of the strain approximated that of the
KAM1 rad1 mutant. When the vector carried the atXPF-1, atXPF-2, or atXPF-3
cDNA, UV-sensitivity of this strain also resembled the KAM1 mutant, indicating no
substantial ability of any of the atXPF cDNAs to complement the rad1 mutation.
Results of the atXPF-3 UV-sensitivity complementation assay are presented
graphically in Figure 3.14. Transcription of the atXPF-3 cDNA under the
complementation conditions used was demonstrated by preparation of RNA from
KAM1/atXPF-3 induction broth culture, synthesis of cDNA, and PCR amplification
with the primer pair AtmRNA-RAD1-F7/R1. A positive amplification result was
taken to indicate production of atXPF-3 mRNA in the cells of the culture. In
addition, the ability of the vector carrying the yeast RAD1 gene to complement the
KAM1 chromosomal rad1 mutation confirms the overall competency of the vector
system.
Overexpression of atXPF-3 from the expression vector does not appear to
inhibit the repair ability of wild-type RAD1 in the yeast NER complex. This is
79
indicated by the normal levels of UV-C resistance displayed in Figure 3.14 by the
wild-type/atXPF-3 strain, and is consistent with the atXPF-3 protein not displacing
RAD1 from its interaction with RAD10. As atXPF-3 is most similar to other RAD1
homologues, it will hereafter be referred to simply as atXPF.
3.12 Assessment of codon useage by atXPD and atXPF
Previous studies have documented the differences between
monocotyledenous and dicotyledonous plants in codon usage. The atXPD and
atXPF cDNAs were assessed for their codon usage, with reference to the
established monocot/dicot patterns, and also the possible implications for expression
level.
In general, codon usage in these cDNAs complied with codon usage in the
Arabidopsis genome overall (Tables 3.5 and 3.6). Specific exceptions to this,
however, were found. For example, in atXPD the GTC, and GTG codons for valine
are under-represented in this cDNA at a percentage frequency of 9%, and 17%
respectively, while the GTT and GTA codons are over-represented at 51% and 23%
respectively. This may be compared to Arabidopsis genome-wide figures of 19%
(GTC), 26% (GTG), 40% (GTT), and 15% (GTA) (refer to Table 3.5). Likewise,
the CCT, CCC, CCA and CCG codons for proline occurr at a frequency of 65%,
7%, 14%, and 14% respectively, compared to genome-wide figures of 38%, 11%,
34% and 17%. Here, as for valine, codons ending in C or G are under-represented in
the atXPD cDNA. atXPD codons for threonine and asparagine also show a similar
pattern. In atXPF, such biasing is not evident at valine and proline codons, although
the GTT codon is still substantially over-represented. Analysis of threonine and
asparagine codons possibly reveals a small bias against codons ending in C or G in
atXPF, but not to the extent seen in atXPD. This may represent a bias, specific to
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the atXPD gene, against those codons ending in either G or C. To further examine
the significance of these observed differences, the atXPD patterns were compared to
data previously used to distinguish between monocot and dicot sequences (Murray
et al., 1989). These authors suggested that monocot and dicot sequences had
intrinsic differences in codon usage for glycine, glutamic acid, aspartic acid and
valine. The characteristics of the atXPD codon usage pattern discussed above are
consistent with, and even amplify these differences. The atXPF codon usage also
strongly conforms to the suggested dicotyledon bias, implying that both atXPD and
atXPF may be dicotyledonous examples of differential codon usage.
It has been suggested, on the basis of codon usage analysis of 2 highly
expressed genes, that such genes are characterised by lower representation of NCG
and NTA codons than is found in broader monocot or dicot samples (Murray et al.,
1989). This decreased representation was quantified by these authors for the codons
GTA, GCG, ATA, ACG, TTA, TCG, CTA, and CCG, which all showed lower
representation in the highly expressed genes than in the monocot or dicot plant
genomes overall. In contrast, neither atXPD nor atXPF showed a reduction in the
occurrence of these codons (Tables 3.5 and 3.6), and so under this criterion are not
expected to be highly expressed genes.
3.13 Isolation of a Rad10/ERCC1 homologue from Arabidopsis thaliana
As discussed in section 1.4.3.1 above, RAD1 functions in NER as a
heterodimer with RAD10. Although an incomplete Lilium longiflorum RAD10
homologue has been reported (Xu et al., 1998), as discussed in section 1.4.3.3, no
full-length Arabidopsis RAD10 homologue is yet known. Retrieval of such a
homologue is naturally a next step in the definition of NER in Arabidopsis, and
higher plants generally. More specifically, the availability of the atERCC1 cDNA
81
may be of assistance in yeast complementation studies using cDNAs for the plant
heterodimer rather than either cDNA alone. Use of the lily ERCC1 sequence for this
task is unwise on the basis that firstly, it is only a partial cDNA, and secondly the
differences between dicot and monocot sequences may render any results
inconclusive.
An A. thaliana chromosome III database entry (Accession number
AC009177) includes a sequence suggested to be a putative repair protein on the
basis of sequence homology. This work is unpublished, however the sequence
identified correlates with our own putative Arabidopsis RAD10 homologue,
retrieved via BLAST (Altschul et al., 1990; Gish and States, 1993) searches using
both the Lily and human ERCC1 proteins as query sequences. Figure 3.15 displays
a protein alignment of RAD10/ERCC1 homologues, including both the lily and
predicted putative Arabidopsis sequences, and is discussed further in section 4.4.
3.14 Isolation of an A. thaliana partial cDNA homologous to RAD2/XPG
Previous sections have described the isolation and characterisation of a
RAD1/XPF homologue and a RAD10/ERCC1 homologue from A. thaliana. As
discussed in the Introduction, these two proteins are thought to combine in a
heteromeric protein complex to form an endonuclease responsible for the incision 5’
to the lesion in NER. To complete the removal of the damaged strand, incision 3’ of
the lesion obviously must occur. In yeast and mammals this incision is made by
RAD2/XPG (O'Donovan et al., 1994), however no plant homologue of this protein
or the related gene is known.
Virtual screening of the A. thaliana database via the TBLASTN search
facility (see section 2.8) (Altschul et al., 1997) using either the S. cerevisiae RAD2
protein sequence or the human XPG protein sequence as the query sequence
82
resulted in retrieval of the A. thaliana genomic clone AB028616. The search
alignment also indicated that the START codon for the putative plant protein would
be at base 24,968 of the genomic clone. By use of the NetGene2 WWW server
(http://genome.cbs.dtu.dk/htbin/nph-webface), a prediction of the intron/exon
processing of the region encompassing 9,000 bp downstream of this position was
attempted. Unfortunately, prediction from this sequence resulted in only limited
useful information in that many of the intron/exon boundaries predicted were left
open-ended, i.e., were not paired. However, manual scanning of the genomic
nucleotide and corresponding amino acid sequence identified a potential STOP
codon at bases 30,981-30,983, which when translated is immediately preceded
in-frame by the amino acid sequence PGRSKKTK. This sequence is similar to the
final eight amino acids of the conserved C-region of RAD2 (GKLKKRKM). This
region of the atXPG nucleotide sequence can also be translated to the equally
similar amino acid string RKRNKKK by moving the reading frame by 1 base.
Consequently, nucleotides 30,964-30,983 were chosen as the farthest 3’ sequence
recognisable in the genomic clone AB028616 from homology comparisons.
3.14.1 PCR amplification and cloning of atXPG partial cDNA
PCR primers were designed to amplify between genomic bases 24,968 and
30,983 from cDNA. These primers are listed in Table 2.1. Primer
AtmRNA-RAD2-F1 binds at the 5’ end of the cDNA, straddling the START codon.
Primer AtmRNA-RAD2-R1 binds to the extreme 3’ end of the cDNA, and includes
a putative STOP codon. Primers AtmRNA-RAD2-F2 and AtmRNA-RAD2-R2 both
bind at base pairs 4,332-4,348 of the cDNA, but in opposite orientations. Synthesis
of these primers allowed PCR amplification of the putative atXPG cDNA
homologue in two fragments, and subsequent sequencing and cloning of the smaller
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fragment. Cloning of the larger fragment is still underway. The 852 bp cDNA and
corresponding amino acid sequence constructed thus far are presented in Figure
3.16. In an alignment with other RAD2 homologues (Figure 3.17) this protein
sequence shows nearly 22% identity, and 46% identity or conservative replacement.
The reported helix-loop-helix (HLH) motif reported from human XPG and thought
to be characteristic of structure-specific nucleases (Park et al., 1997), is also
potentially present in atXPG. The first seven amino acids shown in Figure 3.16
(EAEAQCA), are identical to the final seven amino acids of the HLH motif
(ESQELLRLFGIPYIQAPMEAEAQCA). Although yet to be confirmed
experimentally, the predicted translation of the nucleotide sequence immediately
upstream from this point in atXPG, is: ECQELLQIFGIPYIIAPMEAEAQCA. In
this specific, characteristic motif, the identical residues total to 84%, lending further
weight to the notion of homologous function.
3.15 Translesion synthesis genes from A. thaliana
Translesion synthesis appears to be a process present in organisms as diverse
as bacteria and mammals, and yet no TLS component has been published from a
plant genome. As there is no evidence to suggest that the processes of DNA
replication and also DNA damage repair by photoreactivation, BER and NER are
much more efficient in plants, there probably remains a requirement for TLS in this
group of organisms. Consequently the search for plant TLS genes is a valid quest.
3.15.1 Isolation of a RAD30/XPV homologue from Arabidopsis thaliana
An initial screening of the Arabidopsis database via the search facility
TBLASTN (see section 2.8) using the 713 amino acid human RAD30 protein as
probe resulted in the retrieval of two identical Arabidopsis genomic clones
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(Accesion Numbers AB016874 and AC002342). Both clones apparently contain the
same sequence from chromosome 5, but in reverse orientation. The second genomic
clone includes a protein link (Accesion Number g2660675) detailing a conceptual
translation of this genomic clone into a protein sequence. A protein alignment of
human Pol η and the putative A. thaliana protein (atRAD30) is presented in Figure
3.18. As can be seen, large areas of the alignment show complete conservation, and
in fact of 713 amino acids present in human Pol η, 226 are identical in atRAD30
(30.9%), and a further 241 are conservatively replaced (33.9%) to give a total of
64.8% similarity.
A second protein translation on the basis of intron/exon prediction from the
NetGene2 WWW server largely confirmed the first, and assisted in designing
homologous PCR primers to amplify the putative atRAD30 from a cDNA pool. The
primers synthesised are listed in Table 2.1. These primers are now in use as the
starting point of a subsequent project aimed at retrieving and characterising a
RAD30/XPV homologue from A. thaliana.
3.15.2 Isolation of a REV3 homologue from Arabidopsis thaliana
Identification of an Arabidopsis REV3 homologue was initiated by probing
the Arabidopsis nucleotide database, via the TBLASTN (Altschul et al., 1990; Gish
and States, 1993) (see section 2.8) search facility, using the mouse REV3 protein as
the query sequence. This search returned a nucleotide database entry whose
predicted translation showed significant similarity to the mouse REV3 probe
sequence. The Accession Number of this sequence, a BAC clone constructed from
chromosome 1 of the Columbia ecotype of A. thaliana, is AC004393. This database
entry includes a predicted protein translation sequence for this putative A. thaliana
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REV3 homologue (patREV3). An alignment of this sequence, along with human,
mouse and yeast proteins is presented in Figure 3.19.
The large variation in the size of these homologous proteins is immediately
noticeable, in fact the human sequence is about twice the length of the yeast
sequence (3,130 amino acids vs. 1,504 amino acids). Closer inspection, however,
reveals that this extra length is contained in an amino terminal extension of some
1,600 amino acids, although other authors have proposed a region of amino terminal
sequence homology followed by an intervening unrelated region (Gibbs et al.,
1998). Much greater homology is found when areas further downstream in these
proteins are examined. The human protein has previously been shown to include six
conserved sequence motifs (I to VI) characteristic of DNA polymerases (Gibbs et
al., 1998) which would also be expected to be present in an Arabidopsis REV3
homologue. Examination of these regions in patREV3 reveals striking similarity in
all six regions, lending credibility to the notion of homologous function.
Furthermore, the exonuclease I region identified in the yeast replicative
polymerases (Blanco et al., 1992) and required for the proofreading function of
these proteins, does not appear to have a counterpart in the patREV3 protein,
consistent with the relaxed fidelity required for TLS.
Investigations are currently underway in our laboratory to retrieve and fully
characterise the patREV3 cDNA, using PCR based on the primers for amplification
and sequencing shown in Table 2.1.
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4 Discussion
The previous sections of this document have summarised the current body of
knowledge of plant DNA damage repair and tolerance, particularly NER, and
described the original isolation and characterisation of four genes, two complete
cDNAs, and two partial cDNAs suggested to code for plant homologs of yeast and
mamalian NER components. In addition, two genes coding for putative translesion
synthesis polymerases have been identified, and their cloning initiated. The
following section critically analyses these new plant homologues for the presence or
absence of sequence features relating to their proposed functions. Also, the broader
view of the way in which repair and tolerance of lesions may be linked to cell cycle
progression is discussed.
4.1 Evidence for a homologous role for atXPD
4.1.1 RAD3/XPD mutations affecting repair
Substitutions at a number of positions within the human XPD gene have
been characterised, some of them having no adverse effect phenotypically (Shen et
al., 1998), whilst others confer specific phenotypic effects reflecting loss or
reduction of function in transcription or NER. Likewise in yeast, functional
deficiencies have been attributed to specific mutations. In an attempt to further
define regions of structural importance (as indicated by conservation), and also to
reinforce the notion that atXPD is justifiably a RAD3 homologue, sequence changes
in yeast and human mutants were examined in relation to the predicted atXPD
protein (see Figure 3.3, following page 62).
A G·C→A·T substitution at nucleotide position 706 of the RAD3 gene
resulted in a substitution of lysine for glutamic acid, thus creating the rad3-1
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mutant. This substitution is at codon 236 within the invariant DEA sequence of the
so-called ‘DEAD’ box (helicase motif IV) (Deschavanne and Harosh, 1993;
Friedberg et al., 1995), and resulted in a phenotype that was viable but deficient in
excision repair (Naumovski et al., 1985). Consistent with these observations, the
atXPD protein maintains a glutamic acid residue at the corresponding position.
Likewise, the rad3-2 mutant is defective in DNA excision repair but is
viable. The rad3-2 mutation consists of a G·C→A·T transition at nucleotide position
1381, resulting in a substitution of arginine for glycine at amino acid position 461
(Naumovski et al., 1985). This substitution is within the wild-type invariant ITSGT
sequence. These mutants are thought to be defective in the incision step of repair
(Sung et al., 1988). In common with all other wild-type RAD3/XPD homologues,
atXPD also has a glycine residue at this position.
Point mutations resulting in an amino acid substitution within the conserved
nucleotide binding region resulted in abolition of ATPase and helicase activities, but
surprisingly at the time, did not cause inviability (Sung et al., 1988). The mutant
rad3 Arg-48 has a nucleotide substitution resulting in the change of lysine-48 to
arginine, within the ‘Walker-type’ motif found in other helicases (Friedberg et al.,
1995). This substitution did not affect the ability of RAD3 to bind ssDNA or ATP,
however it did abolish the ATPase activity and the related helicase activity (Sung et
al., 1988; Coin et al., 1998), implying that the helicase activity is not required for
the essential role of RAD3 in the TFIIH complex. In contrast, the helicase activity
of ERCC3 (the human homologue of RAD25) is apparently absolutely required for
viability (Ma et al., 1994), and the rad25 Arg-392 mutant (an ATP-binding region
mutation) is extremely NER deficient (Sung et al., 1996). rad3 Arg-48 mutants are
highly UV sensitive, because although the rad3 Arg-48 mutant can apparently
89
initiate incision of DNA damaged by UV light, it is unable to complete the repair by
removing pyrimidine dimers from the DNA (Sung et al., 1988). The NER efficacy
of the rad3 Arg-48 mutant is virtually abolished (Sung et al., 1996). Nevertheless
these mutants are viable. A second rad3 mutation identified at this position
(rad3-21) in which the lysine is replaced by a glutamic acid, is also extremely UV-
sensitive (Song et al., 1990). In Arabidopsis, atXPD maintains a lysine residue at
position 48.
The rad3-101 and rad3-102 mutants are two other yeast strains in which the
mutations responsible have been characterised. These two mutations are not lethal
and do not confer markedly increased UV sensitivity, but instead comprise the
so-called rem mutations, a class in which increased spontaneous recombination and
mutation is seen. rad3-101 is a G⋅C → A⋅T transition at nucleotide 708 resulting in
a threonine for alanine substitution at codon 237 within the “DEAD box”.
Intriguingly, this mutation is in the codon next to that in which the rad3-1 mutation
occurs, conferring greatly increased UV-sensitivity. Possibly the threonine/alanine
substitution of rad3-101 is less disruptive than the substantial change in character
associated with the glutamic acid/lysine substitution of rad3-1. Again, the
Arabidopsis protein conforms to the consensus sequence of this conserved motif by
maintaining an alanine residue at this position. The rad3-102 mutation was found to
be a C⋅G → A⋅T transversion at nucleotide 1981, resulting in tyrosine instead of
histidine at codon 661 (Montelone and Malone, 1994). The specificity of the
rad3-102 mutator effect may be an indication of the influence of this gene on
recombination or postreplicative mismatch repair (MMR) (Montelone and Malone,
1994). Other evidence linking RAD3 and MMR includes the observation that the
efficiency of mismatch correction is enhanced by about 25% in a rad3-1 mutant
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strain (Yang et al., 1996). This increase was apparently not attributable simply to
increased transcription of mismatch repair genes. Surprisingly, atXPD is the only
protein sequence of those considered varying at position 661 (the rad3-102 site),
having a glutamine residue at this position in contrast to the otherwise perfectly
conserved histidine. It should be noted that a single base change at position 3 of the
codon is capable of causing the change of histidine to glutamine (CAC or CAT to
CAA). Sequencing of this region of selected atXPD cDNA clones in both directions
revealed only CAA or TTG, depending on the direction of sequencing. In addition
the database genomic sequence also codes for glutamine. Perhaps the requirement at
position 661 is actually for a hydrophilic amino acid such as histidine or glutamine,
rather than a somewhat more hydrophobic residue such as tyrosine, although the
consensus retention of histidine seemingly argues against this. Alternatively, the
plant sequence may simply be the result of evolutionary divergence.
An additional compilation of other mutations in RAD3, and their effect on
UV-sensitivity and mutation rate (Song et al., 1990), provides further opportunity to
evaluate the functional significance of individual amino acids in the Arabidopsis
atXPD protein sequence. The rad3-20 allele carries a glycine to aspartic acid
substitution at codon 47, resulting in severe UV-sensitivity and an increase in
spontaneous mutation rate of several orders of magnitude. Again, Figure 3.3
confirms that glycine is found at this position in all the homologues compared,
including that from Arabidopsis. The rad3-24 mutation is a glycine to arginine
substitution at codon 604 in the VIIb helicase domain, conferring UV-sensitivity
and elevated spontaneous mutation rate. At this position also, atXPD complies with
conservation of the glycine residue. A double mutation composed of a threonine to
isoleucine change at codon 462, and a serine to leucine change at codon 464 was
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detected as the rad3-26 allele. It results in moderate UV-sensitivity, not surprising
as these are the amino acids next to, and third away from, respectively, that
substituted in the rad3-2 mutation. The atXPD protein shows perfect conservation at
both positions. The rad3-28 allele is also a double mutation, at codons 543 (serine
to leucine) and 594 (arginine to cysteine). The rad3-28 mutant is only moderately
UV-sensitive, and has a spontaneous mutation rate only several fold higher than
wild-type. Again the atXPD protein is perfectly conserved at these positions within
the VIIb helicase region. The rad3-32 mutant showed moderate UV-sensitivity and
resulted from a substitution of serine by phenyalanine at codon 209. This also is at a
position at which serine is found in the homologues considered, including atXPD.
The XPD homologue from Arabidopsis also terminates in protein
alignments 20 amino acids before the yeast protein carboxyl terminus, as does
human XPD, thereby omitting the highly acidic carboxyl terminal tail. This acidic
region of the yeast protein is in fact not required for viability, although its influence
on UV repair is unknown (Reynolds et al., 1985).
In summation, of the twelve specific sites in yeast RAD3 at which
substitutions are known to have a phenotypic effect on repair, atXPD is completely
conserved at eleven, and shows conservative replacement at another. Even if
considered in isolation, this is persuasive of homologous function. An even broader
assessment can be achieved however, if mutations affecting viability are included.
4.1.2 RAD3/XPD mutations affecting viability
The rad3-G595R mutant exhibits a temperature-sensitive growth phenotype,
resulting from the substitution of glycine by arginine in the VIIb helicase region.
The mutant strain has a doubling time about 1.8 times longer than the wild-type at
30oC, and 5.3 times longer than wild-type at 37oC. This amino acid substitution
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results in an elevated level of recombination, apparently independent of NER (the
spontaneous mutation rate and UV-sensitivity of this mutant strain are not
significantly different to that of the wild-type) (Maines et al., 1998), and is
reminiscent of the rad3-101 and rad3-102 rem mutants. The plant protein has a
glycine residue at the corresponding position, in common with all other
homologues. It is interesting to note that one of the two substitutions responsible for
the moderate UV-sensitivity of the rad3-28 mutant was at codon 594, adjacent to
the temperature-sensitive rad3-G595R viability mutant.
The rad3ts-1 mutant also displays a temperature-sensitive growth habit
(Naumovski and Friedberg, 1987), and carries a G⋅C → Α⋅T transition at position
218 (codon 73), resulting in serine substituted by phenylalanine. This serine residue
is conserved in all RAD3 homologues except atXPD, in which it is conservatively
replaced by threonine, and is flanked by 3 or 5 conserved or conservatively replaced
amino acids on either side. Clearly, this is a functionally significant region, beyond
the importance to secondary structure of the included cysteine residue. The doubling
time of the rad3ts-1 mutant in liquid culture at 30oC is 1.6 times that of the isogenic
wild-type, and at 37oC only 2-4 doublings occur. The mutant strain is also slightly
UV-sensitive compared to the wild-type, implying some minor duality of function
for this region of sequence.
The third temperature-sensitive rad3 mutant to be considered is the rad3ts14
strain, which includes a single point mutation in codon 590 of rad3 causing a
substitution of cysteine by tyrosine (Guzder et al., 1994). This codon is within the
helicase VIIb region. All homologues, including atXPD, maintain a cysteine at this
position. This combination of cysteine conservation and temperature sensitivity hint
at this mutation disrupting or destabilising the tertiary structure of the wild-type
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yeast protein, and thereby exerting an effect on transcription. If this is the case, a
mutation causing a substitution of another cysteine residue in the RAD3 protein,
normally linked to the cysteine at residue 590 by a disulphide bridge, should have
no temperature-sensitivity effect above that seen with the rad3ts14 allele. The NER
status of this strain has not been determined, although mutations at nearby sites have
been assessed for their impact on NER. For example, the rad3-G595R mutation
which imparts a temperature-sensitive growth habit but approximately wild-type
UV-resistance (as discussed above) is only 5 amino acids away from the rad3ts14
mutation. The rad3-28 mutant carries two mutations (see above), one of which is
four residues away from the rad3ts14 mutation, and adjacent to the rad3-G595R
mutation. This mutant is moderately UV-sensitive, and is apparently unaffected for
viability, but because of the presence of two mutations it is difficult to distinguish
the functional consequences of each. Overall, these mutations in the VIIb helicase
domain are certainly capable of affecting viability, and perhaps able to impact on
UV-resistance. Unfortunately, the UV-sensitivity imparted by the rad3ts14 mutation
is not known.
Human studies also have investigated the relationship between specific point
mutations in the XPD gene and clinical consequences. Trichothiodystrophy (TTD)
is a condition featuring physical and mental retardation, brittle hair, and
sun-sensitivity. In contrast to those suffering xeroderma pigmentosum (XP), TTD
patients are not generally cancer-prone (de Boer and Hoeijmakers, 2000). Both
conditions involve mutations in the human XPD gene, it being suggested that XP
results from repair deficiency and TTD from effects on transcription (Taylor et al.,
1997; de Boer and Hoeijmakers, 2000). Mutations within the carboxyl terminus
region generally of XPD have been credited with a reduction in the binding of p44
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to XPD in the transcription initiation function of TFIIH (Taylor et al., 1997; Coin et
al., 1998). These mutations were largely localised to two overlapping regions of the
human XPD gene: those coding for amino acids 675-730 in the XP-D syndrome,
and 713-730 in the TTD syndrome (Coin et al., 1998). Within these two regions, the
atXPD protein shows high sequence conservation, and is significantly more similar
to the human and fish proteins than to the S. cerevisiae and S. pombe sequences. In
particular, the conserved motif within the TTD domain of FLRXMAQP varies at
only one amino acid across all the aligned sequences (representing mammal, yeast,
fish and plant). It is interesting to ponder the phenotype of a TTD-type mutant in a
plant (or even yeast), although very difficult to predict the impact of the
modification of a process as fundamental as transcription.
Of the human XPD mutations mapped, only those resulting in changes at
amino acid positions arginine-683 (found in XP only), arginine-616 (found in both
XP and TTD), arginine-722, arginine-112 and arginine-658 (all found only in TTD)
will be considered here (due to the clear characterisation of these mutations) (Taylor
et al., 1997). If the premise of XP and TTD arising from repair or transcription
defects, respectively, is accepted, then some indication of the protein regions
specifically required for those functions is given. From this, several conclusions can
be reached. Firstly, atXPD shows complete conservation of these residues at each
position, thus emphasizing the striking conservation found in crucial areas and
reinforcing the validity of the proposed homologous function of atXPD. Secondly,
in the light shed by the Arabidopsis data in conjunction with yeast and human
sequences, two regions seemingly essential for viability appear. The first region
includes that altered in the yeast rad3ts-1 mutation and the human R112 TTD
mutation and encompasses the conserved helicase II motif (amino acids 74 to 112).
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The second region extends from the rad3ts14 mutation perhaps to the carboxyl
terminus of the proteins (amino acids 590 to 760), and so includes both the XP and
TTD regions suggested above (675-730 and 713-730) (Taylor et al., 1997; Coin et
al., 1998).
4.2 atXPD is an Arabidopsis RAD3/XPD homologue.
By probing the available Arabidopsis database sequences, an Arabidopsis
homologue of RAD3/XPD has been identified, and its corresponding complete
cDNA cloned and characterised, including the 3’-untranslated nucleotide sequence
to the mRNA poly-A tail (sections 3.2 and 3.3). The predicted plant protein
maintains conservation of amino acid residues previously shown to be required for
NER and the consequent wild-type UV-resistance (section 4.1.1).
In addition, inspection of the many amino acid residues now demonstrated to
be specifically required in RAD3 or XPD for wild-type transcription function and so
viability, shows overwhelming conservation of the corresponding residues in atXPD
(section 4.1.2). This conservation is much higher than would be expected from
chance alone, and together with conservation related to repair, adds considerable
support to the notion that the Arabidopsis atXPD gene codes for a protein that is
indeed a homologue of the yeast RAD3 and human XPD proteins, involved in both
NER and the transcription factor TFIIH. Possible phylogenetic relationships of
these proteins are depicted in Figure 4.1, and show the plant sequence as clearly
distinct from the fungal and vertebrate sequences (which have all been arranged as
expected).
Although experimentally the atXPD cDNA could not complement a yeast
rad3-1 mutant for UV-resistance, it was able to significantly restore viability at
39oC to a strain carrying a temperature-sensitive rad3 allele (rad3ts14). A previous
96
study has reported that the human XPD cDNA was able to restore viability but not
UV-resistance to a Saccharomyces cerevisiae rad3 deletion mutant (Sung et al.,
1993). Observed differences in growth rates of the rad3∆ strain carrying RAD3 or
human XPD were attributed by these authors to differences in the efficiency with
which each protein could participate in the essential RAD3 function. An estimate of
this relative efficiency was made by measurement of the growth rate in nutrient
broth culture of otherwise isogenic haploid yeast strains carrying either human XPD
or RAD3. XPD conferred a doubling time of about 7 hours whereas RAD3 allowed a
doubling time of about 1.5 hours, i.e., more than 4 times faster. From data generated
during the current investigation, the ratio of the minimum doubling times in
expression medium broth at 39oC of D5225∆’32-38A with pRS95 and
pYES2.1HIS-Topo-V5 carrying RAD3, compared to D5225∆’32-38A with pRS95
and pYES2.1HIS-Topo-V5 carrying atXPD was 0.86 times (see Figure 3.6). The
similarity of these generation times indicate that in the system used, atXPD and
RAD3 are similar in their ability to promote culture growth (cell division) in the
presence of the rad3ts14 allele.
In contrast, mutant human XPD genes incorporating either a substitution of
arginine by tryptophan at codon 722, or a frameshift-induced premature termination
signal were unable to complement a rad3∆ strain for viability (Guzder et al., 1995).
Wild-type budding yeast, fission yeast and Arabidopsis RAD3 protein homologues
all have an arginine residue at the position equivalent to codon 722 of human XPD.
The rad3ts14 strains complemented by either RAD3 or atXPD each grow to a
maximum cell density at 39oC of about 8x106 cells/ml. These strains grow to
maximum cell densities at 30oC of about 2.5x107 cells/ml, approximately three
times greater. This could be a reflection of an inherently reduced strain viability at
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39oC, unrelated to RAD3. Alternatively however, it may be a consequence of
competition for incorporation into functional protein complexes (for example TFIIH
but also perhaps others) between the thermally-denatured rad3ts14 protein and the
wild-type RAD3 or atXPD proteins. In relation to this, results shown in section 3.6
indicate that when the wild-type RAD3 gene is available, (even when carried on the
galactose-inducible multicopy plasmid), the rad3ts14 allele is dispensable for
viability. In contrast, the plasmid carrying the rad3ts14 allele apparently could not be
lost from the atXPD-complemented strain, without loss of viability. Since both the
rad3ts14 and atXPD sequences are apparently required for viability, this argues that
each sequence is coding for a seperate essential function. It may be that whilst the
atXPD cDNA is able to enhance the viability of the ts mutant, it may not be able to
complement for all functions of RAD3, and so the rad3ts14 allele cannot be lost
even in the presence of atXPD if viability is to be retained.
Two inferences emerge from the possibility that atXPD does not fully
complement the absence of RAD3. Either rad3ts14 and atXPD can together complete
the RAD3 aspect of the TFIIH complex, or alternatively, that the two proteins can
each fulfill a viability requirement, either in the TFIIH complex or in some
unknown function. The first possibility requires that either the TFIIH complex
contains at least two copies of RAD3, an option not supported experimentally, or,
that the TFIIH complex is dynamic in structure and is continually disintegrating and
reforming, so allowing interchange of rad3ts14 and atXPD at steps of the
transcription process for which each is functional. At present, there appears to be
little experimental evidence to support this. The second option is that there are
actually at least two distinct viability functions of RAD3, only one of which can be
performed by rad3ts14 at the restrictive temperature, and the second by atXPD. In
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the case of the rad3ts14 protein, this residual function must be distinct from the
activity disabled by the temperature-sensitive mutation (and which is now
performed by atXPD). This scenario of dual essential functions of the RAD3 protein
is consistent with competition between rad3ts14 and atXPD or RAD3 resulting in a
reduction of fitness and consequent lower final cell titre at 39oC compared to 30oC.
While no conclusive evidence exists to support the possibility of two
essential functions, some link to the rad3-102 (Montelone and Malone, 1994) yeast
mutation may exist. As previously described (see section 4.1), this mutation
phenotypically results in wild-type viability and moderate UV-sensitivity, but an
elevated frequency of mitotic recombination, an unusual phenotype compared to
those conferred by most rad3 mutant alleles. The link to atXPD lies in the specific
sequence change constituting this mutation. Wild-type RAD3 has a histidine residue
at amino acid position 661 whereas the rad3-102 mutant has a tyrosine, an amino
acid replacement apparently not impinging on viability, despite the impact on
UV-sensitivity. The atXPD protein has a glutamine substitution, the only wild-type
sequence in this study found to vary from histidine at the corresponding location,
This is the only location at which a substantial deviation away from the consensus
amino acid sequence is detected in atXPD. If this deviation is significant, it may
carry the implication that specific amino acids in this region are important for a
viability function in yeast, and so perhaps explain the inability of atXPD alone to
substitute for RAD3.
The plant atXPD protein, in common with vertebrate homologues, omits the
carboxyl terminal tail found in the corresponding proteins from both Saccharomyces
cerevisiae and Schizosaccharomyces pombe. The effect of this omission on the
activity of this protein in yeast NER is unknown, although this may be another
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explanation of the similar failures of plant and vertebrate homologues in
complementation of complete viability or repair functions.
4.3 atXPF is an Arabidopsis RAD1/XPF homologue
Another step in the investigation and definition of NER in plants, as
represented by the model dicot Arabidopsis thaliana, has been taken with the
cloning of a plant XPF/RAD1 homologue, as described in this report and confirmed
by others (Liu et al., 1999; Gallego et al., 2000). This includes an assessment of the
efficiency with which this plant coding sequence can complement for UV repair in
vivo a large deletion in the chromosomal yeast RAD1 gene.
As a familiar, well-characterised model organism, S. cerevisiae is
well-suited to this type of comparative study. Given the phenotypic consequences of
inactivation of RAD1 and RAD1 homologues, a plant RAD1 mutant would be
expected to be viable, UV-sensitive, and potentially deficient in recombinational
repair pathways, perhaps also including Agrobacterium transformation. In fact, the
uvh1-1 Arabidopsis mutant recently characterised (Liu et al., 2000), and now known
to carry a mutation in the homologous gene, is hypersensitive to both UV-radiation
(seen as the yellowing and shriveling of exposed tissue) and ionising radiation
(indicated by the failure of true leaves to develop normally) (Harlow et al., 1994).
Structurally, the yeast RAD1 protein has been partially characterised. A
bipartite nuclear transport motif is assigned to amino acids 517 onwards (Friedberg
et al., 1995). This yeast motif is poorly conserved in the Arabidopsis protein. The
RAD10 binding region is thought to be between amino acids 809 and 997 of RAD1
(Bardwell et al., 1993). Figure 3.13 displays a protein alignment including RAD1
and atXPF, and shows that within this region, areas of significant amino acid
conservation exist between RAD1 and atXPF. Of the 188 amino acid residues in
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RAD1 thought to be involved in binding to RAD10, 73 locations in atXPF are
occupied by the identical amino acid, and a further 63 show conservative amino
acid replacement, giving overall homology within the putative RAD10 binding
region of 136 of 188 amino acids (72%). Of the non-conserved positions, the
longest run is within the section of sequence bounded by residues 914 and 936 of
RAD1, where 14 amino acids (ERRNYKNKDISTVH) are missing from the atXPF
sequence. Furthermore, these amino acids are also largely missing from all of the
vertebrate and insect sequences aligned, but perhaps present in the RAD1
homologue from the filamentous fungus Neurospora crassa. The possibility that the
absence of these residues in atXPF precludes a satisfactory interaction with yeast
RAD10 in the UV complementation studies cannot be dismissed.
The recently reported UVH1 and atRAD1 cDNA sequences (Gallego et al.,
2000; Liu et al., 2000) match very closely the atXPF sequences, and confirm the
results of the present study, in terms of both nucleotide sequence and gene structure.
Disparity between the sequences is restricted to the deviations imposed by the
variable transcript processing seen in atXPF species, and a single base substitution
at nucleotide position 1,251, at which atXPF-1, 2, and 3 have a G, whereas UVH1
has an A (Figure 3.9). This transition results in a glycine residue in the atXPF
protein, and an aspartic acid residue in the UVH1 protein. The atRAD1 protein also
appears to have an aspartic acid residue at this position (Gallego et al., 2000).
Nonetheless, these cDNAs are obviously specifying virtually identical proteins.
Unfortunately, at the time of this writing, the Genbank Accession Number provided
by Gallego et al. (2000) is inactive, and so the complete nucleotide sequence of their
atRAD1 cDNA is effectively not available.
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The mutation responsible for the uvh1-1 mutant phenotype was found to be a
second G to A transition 8 bp upstream of the 5’ end of exon 6 in the genomic
sequence (Liu et al., 2000). This change was suggested to modify the intron/exon
processing in the region, and so result in the inclusion of a premature STOP codon
in the corresponding mRNA. This STOP codon would result in a truncated protein
lacking the carboxyl terminal region thought to be required in yeast for interaction
with RAD10. Interestingly, this region is one of the areas of variability between
atXPF-1 or 3 and atXPF-2, being present only in the atXPF-2 cDNA (Figures 3.7
and 3.8). Intron/exon cleavage at this site specifically, however, is unique to uvh1-1.
In total, this indicates that there are at least four possible transcripts for atXPF, at
least one of which (uvh1-1) imposes a demonstrated phenotypic penalty.
Such variation in transcript processing has been previously reported for
other plant genes. For example, this process is known to be responsible for the
production of two rubisco activase polypeptides in spinach and A. thaliana, in
which the processing of an intron at the 3’ end of the RNA determines which of the
two polypeptides is produced (Werneke et al., 1989). In addition, variable
processing of the U1-70K transcript from A. thaliana results in two distinct
mRNAs, one of which retains a region spliced out of the other (Golovkin and
Reddy, 1996). The product of only one transcript is recognised functionally.
Similarly, variable processing of the A. thaliana FCA transcript also produces
multiple polypeptides, of which only one appears to be full length and functional
(Macknight et al., 1997).
Variable transcript processing can also influence gene expression. For
example, retention of introns can increase levels of steady state RNA of reporter
genes, and reporter gene product activity (Luehrsen and Walbot, 1991).
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Furthermore, intracellular targeting of proteins may be affected. Secreted or
membrane-bound ceruloplasmin in mammals is produced from the same hnRNA,
but spliced differently at one intron to result in the presence or absence of a
membrane anchor sequence (Bharatkumar et al., 2000). The report of the atXPB
cDNA (homologous to RAD25) during this investigation coincidentally also
described variable processing, resulting in a cDNA containing two residual
unspliced introns (Ribiero et al., 1998). Whether such processes underly the
multiple transcripts of atXPF is as yet unknown, but the possibility cannot be
discounted.
atRAD1 was reported to be functional in the partial complementation of an
S. pombe rad16 UV-sensitive phenotype. At a dose of approximately 40 J/m2 the
surviving fraction increased from about 3% for the mutant strain to about 20% for
the mutant strain carrying atRAD1, at 80 J/m2 the surviving fraction increased from
about 0.1% for the mutant to 1% in the complemented mutant (Gallego et al., 2000).
Wild-type survival at 80 J/m2 appeared to be about 80%. Despite being the best
sequence match with other RAD3 homologues of the three atXPF species
characterised (see section 3.8), atXPF-3 cannot substitute for RAD1 in the in vivo S.
cerevisiae UV-sensitivity assay employed in this study (and nor can atXPF-1 or
atXPF-2, see section 3.10.1). Whether this is due to loss of elements of the
RAD1/RAD10 binding domain, the absence of a yeast nuclear targeting sequence in
atXPF-3 or simply to structural divergence remains to be determined. Since the
KAM1 rad1 mutant was constructed by deleting about 70% of the 5’ RAD1 coding
sequence (bases 476 to 2600) (Kunz et al., 1990), it is unlikely, although not
impossible, that atXPF-3 is simply out-competed for the native RAD1 binding sites
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by any truncated RAD1 still expressed (the RAD10 binding site is coded for by
bases 2419 to 2979).
As to why complementation might be seen in a S. pombe assay but not in an
S. cerevisiae assay, the answer may lie in the greater overall similarity of the S.
pombe and Arabidopsis proteins compared to the S. cerevisiae and Arabidopsis
proteins, thus providing an increased chance of a positive substitution of one for the
other. Indeed, the observations of Gallego et al. (2000), regarding partial
complementation of an S. pombe mutant by the Arabidopsis RAD1 homologue
(atRAD1) may have bearing on the consequence of the absence of the 14 amino
acids from the RAD10 binding region, as discussed above. The S. pombe RAD1
homologue (RAD16) is also missing these residues, and so the absence of these
residues in atRAD1 would not necessarily preclude a successful interaction with the
S. pombe RAD10 homologue. Alternatively, perhaps the A/G single base
substitution at base number 1261 in atXPF compared to atRAD1 is impacting on the
activity of the plant protein, irrespective of the species in which the assay is
performed.
Figure 4.2 displays a possible phylogenetic tree of the relationship between
RAD1 protein homologues, including atXPF-3, as calculated by the CLUSTALW
multiple alignment program. Interestingly, atXPF-3 is shown as most closely related
to the fungal homologues, rather than the vertebrate sequences. Confidence in the
tree may be drawn from the arrangement of the two vertebrate sequences together,
the two fungal sequences together, and the distinct radiation of the insect entry.
4.4 Evidence for a homologous role for atERCC1
Perusal of Figure 3.15 reveals that amino acid conservation in the
amino-terminal region of the aligned RAD10 homologues is not high, there being
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non-conservative replacement across the alignment at 88 positions corresponding to
the first 90 amino acids of RAD10. Mutational analysis studies revealed that the
first 91 amino acids of human ERCC1 are dispensable for activity in NER or
recombination (Sijbers et al., 1996). The human sequence from amino acids 91 to
119 has been suggested to function as an XPA-interaction domain (Li et al., 1994).
Within the region of the atERCC1 protein corresponding to this domain, 22 of 29
amino acids are conserved or conservatively replaced, although an apparent
insertion of seven additional amino acids partway through this region is as yet
unexplained. The C-terminal region of the alignment encompassing the final 130
amino acids of RAD10 also shows much greater conservation, with 73 of 130 amino
acids conserved, or conservatively replaced, across all proteins in the alignment.
Within this C-terminal region, 8 of the final 12 amino acids of human ERCC1 are
required for resistance to mitomycin C or UV-irradiation (Sijbers et al., 1996), and
in fact, the XPF binding region of the 297 amino acid human ERCC1 protein is
known to encompass amino acids 224 to 297 (de Laat et al., 1998). Within this 73
amino acid sequence atERCC1 shows identity with the human ERCC1 sequence at
35 positions and conservative replacement at a further 12 sites, resulting in a
regional similarity of 64%.
The putative Arabidopsis protein, although somewhat longer than the others,
(perhaps due to errors in the intron/exon predictions) shows a similar level of
commonality in the alignment as do the other members. Comparison of atERCC1
with the human ERCC1 protein alone reveals that of the 298 amino acids in human
ERCC1, 130 (44%) are completely conserved and a further 103 amino acids (35%)
are conservatively replaced. In the region of atERCC1 homologous to the eight
required carboxyl terminal amino acids of human ERCC1, five amino acid residues
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match the human sequence, while there is one substitution of tyrosine for
phenylalanine, one threonine for valine, and one phenylalanine for leucine; all
conservative substitutions. In addition, atERCC1 is perfectly conserved at nine of
ten specific amino acid sites scattered through the human ERCC1 protein from
amino acid number 107 to amino acid number 200, previously shown to have an
influence on either NER, recombination, or both (Sijbers et al., 1996). The tenth
position shows a valine for glutamine substitution, as does the lily protein.
Interestingly, overall, RAD10 from S. cerevisiae is the least similar of the six
sequences compared, a feature perhaps of some impact on the atXPF
complementation assay result of this study.
Identification of this putative Arabidopsis ERCC1 homologue has paved the
way for a subsequent project, currently underway, to PCR amplify, clone and
characterise the complete atERCC1 cDNA corresponding to this protein. This will
allow us firstly to define the atERCC1 homologue, but more importantly to test the
ability of this cDNA to complement an S. cerevisiae rad10 null mutant, and also, to
test the complementation effect of the two plant cDNAs together in either of the
yeast mutants. These experiments may allow us to determine the reason for the
inability of the atXPF-3 cDNA to complement the yeast rad1 mutant for NER.
4.5 Evidence for a homologous role for atXPG
A part of a putative homologue of the human and yeast XPG/RAD2 cDNAs
has been isolated, cloned and sequenced from Arabidopsis. Conservation of the
predicted amino acid sequence in specific functional regions of related proteins can
be a compelling indicator of homologous protein function, and such conservation is
apparent when the putative atXPG protein is compared to recognised RAD2
homologues. For example, a mutation causing a substitution of aspartic acid by
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alanine at residue number 812 in the human XPG protein has previously been
shown to eliminate wild-type incision activity (Wakasugi et al., 1997). This site is
within the atXPG sequence characterised thus far and displays conservation of the
aspartic acid residue found in the human protein. Amino acids 981-1009 of the
human XPG protein are thought to be involved in the direct interaction between
XPG and PCNA. The corresponding sequence in atXPG shows partial conservation
of 57% identity or conservative replacement (see Figure 3.17). This is consistent
with the maintenance of a direct link between incision and DNA polymerisation in
both humans and plants. Intriguingly, in humans this interaction was found to
compete for binding of PCNA with the p21 cyclin-dependent kinase inhibitor
protein (Gary et al., 1997), an intrinsic component of the p53 cell cycle regulation
pathway.
Careful scrutiny of the plant genomic clone translated in the three forward
reading frames reveals striking conservation (identity or conservative replacement)
in regions correlating to the N and I regions of RAD2 (Harrington and Lieber,
1994). The atXPG sequence resembling the N region of RAD2 extends from atXPG
genomic nucleotide number 1 through to number 366, for the I region; from
genomic nucleotide number 4,293 to 5,045, and for the C region; from genomic
nucleotide number 5,999 through to 6,018. Predicted protein translations for these
regions are shown in Figure 4.3 aligned with the corresponding N, I and C regions
of RAD2, mouse XPG, and human XPG proteins. Clearly, significant conservation
of residues exists across the four proteins in the alignment. More precisely, residues
Asp-77 and Glu-791 of human XPG have been shown to be essential for wild-type
NER incision activity (Constantinou et al., 1999), and should be maintained in
functional XPG homologues (see Figure 3.17). atXPG is predicted to display
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complete conservation of both these residues. Notably, atXPG shows a large stretch
of sequence between the N and I regions, consistent with other RAD2 homologues,
and distinct from FEN-1 homologues. In addition the atXPG nucleotide sequence is
clearly different to the FEN-1 homologue recently reported from rice, and lacking
this intervening sequence (Kimura et al., 2000).
Overall, the strong conservation of the N, I and C regions in atXPG, in
conjunction with the conservation of amino acid residues at sites of functional
significance, adds weight to the notion that this Arabidopsis gene codes for a plant
homologue of the S. cerevisiae RAD2 and human XPG NER proteins.
4.6 Coordination of NER in plants
As discussed in section 1.4.3.1, many NER proteins are known to function
as components of a multiprotein complex in yeast and human DNA repair.
Coordination of the expression of several yeast NER genes has previously been
suggested, based on the observation of common motifs in the upstream
non-translated regions of RAD1, RAD2 and RAD3 (Nicolet et al., 1985). These
motifs, designated CS1 and CS2, were found 146 to 160 bp and 289 to 339 bp
respectively upstream from the START codons. Comparisons of the nucleotide
sequences of 1000 bp of the 5’ untranslated regions of the atXPF, atXPD and
atXPG genes reported in this study revealed no such common motifs of any
sequence (alignment not shown). This could mean either that these regions are in
fact not involved in coordination of expression of these genes in yeast or plants, or
alternatively that control in Arabidopsis is regulated by a different mechanism to
that used in yeast.
Generally, pathways of transcriptional control of plant DNA repair genes have
not yet been revealed, nor has any cell cycle mechanism for coordination of repair
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been discovered, although sufficient similarity with other eukaryotic mitotic cell
cycles have been found to encourage investigation.
4.7 NER and cell cycle control linkages in plants
It is intriguing to ponder the details of a putative cell-cycle-coordination
system in plants comparable to that found in mammals, given that a plant p53
homologue probably does not exist. Despite some early Western blot data to the
contrary (Georgiva et al., 1994; Loidl and Loidl, 1996), no plant p53 sequence has
been published, nor do database searches retrieve a convincing homologue. Perhaps
a plant cell, being relatively immobile, and seemingly not prone to systemic
neoplastic transformation, does not require such a vigilant “guardian of the genome”
to monitor cell proliferation. For example, plants in which cell division was
transgenically stimulated or impaired to alter the normal pattern of cell proliferation
showed little morphological consequence, and certainly no sign of tumorigenic
growth (Hemerly et al., 1995; Doerner et al., 1996). It is known however, that
homologues of other mammalian proteins acting downstream of p53 in the human
cell-cycle regulation pathway are present in plants, including atXPD (this study),
XPBara (Ribiero et al., 1998), MSH2 (Culligan and Hays, 1997), PCNA (Park et al.,
1998), and the retinoblastoma protein (Rb) (Grafi et al., 1996). It is also known that
upstream regulators of p53 such as the cyclin D family are represented in the plant
protein repertoire (Dahl et al., 1995; Soni et al., 1995).
Individual plant cells are able to exit or re-enter the cell cycle in either G1 or
G2 phase (Hirt, 1996). In G1 phase there exists a critical point beyond which the
cell is committed to continuing in the cell cycle. This is equivalent to the START
point in yeast, and the “restriction” point in mammalian cells. The key plant
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regulators of transition through G1 phase are D and E type cyclins (Xiong et al.,
1992; Hirt and Heberle Bors, 1996).
In other organisms, cyclin-dependent kinases (Cdk4 and Cdk6) associate
with D type cyclins and phosphorylate the Rb protein, (almost all of which is
phosphorylated before the G1/S transition is reached (Zhang et al., 1992)). This
releases Rb-bound E2F type transcription factors (Sherr, 1996) and so activates
genes required for S phase DNA replication. Direct plant homologues of
mammalian D type cyclins have been found, and were shown to be responsive to
growth factors, hormones and mitogens (Doerner et al., 1996; Hirt, 1996; Hirt and
Heberle Bors, 1996). Both B and D type cyclins have been characterised from
alfalfa, and a study of expression of D type cyclins in Arabidopsis supported the
presumed function of D type cyclins in a retinoblastoma phophorylation pathway
(Fuerst et al., 1996). A and B type cyclins function through G2/M phases, (although
multiple A type cyclins were found throughout the cell cycle in Nicotiana tabacum
(Reichheld et al., 1996)), and E type cyclin concentration increases cyclically
around the START point, implying a function in G1/S transition (Dahl et al., 1995).
Studies of the destruction of mitotic cyclins (B type cyclins) in vertebrates revealed
the covalent attachment of the protein ubiquitin to cyclins destined for degradation.
This “tagging” resulted in a decrease in cyclin concentration and consequent
continuation of the cycle from mitosis into G1 phase (Lodish et al., 1995; Guadagno
and Newport, 1996). A ubiquitin carrier protein resembling RAD6 from S.
cerevisiae has been reported from A. thaliana (Sullivan and Vierstra, 1991).
Given that most cells in seeds are arrested in G1 phase, D and E type cyclin
dependent pathways are expected to be particularly relevant to seed and pollen
viability, as it is this cell cycle phase during which time is required for repair. In
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addition, the totipotency of plant cells (Goldberg, 1988) would seem intuitively to
suggest a requirement for cell-cycle regulation systems at least comparable to those
found in mammals (although animals are susceptible to metastasis and cancer
largely because any tumorigenic cells generated can be transported via the
circulatory system to potentially destroy the entire individual). In total, this
circumstantial evidence for a linkage between DNA repair and cell cycle
progression is compelling, and the apparent absence of a key component (a p53
homologue) central to the process increases the intrigue. The alternative that plants
do not need a p53 type-system would represent a major difference in comparative
cell physiology on both a biochemical and evolutionary level. Either result promises
great interest.
4.8 NER systems in higher plants
Plants are exposed to UV radiation levels as part of the solar flux upon which
they depend for photosynthesis. The primary plant response to UV radiation is to
generate UV absorbing pigments as a protective shield, however this may not be
sufficient, and for other sources of DNA damage this strategy is ineffective. For
example, chemical exposure, ionising radiation, metabolic processes, DNA
replication, and dehydration during pollen and seed production have all been
implicated in loss of DNA integrity. Studies of DNA repair mechanisms in plants
have been infrequent, despite the consequences of plant DNA repair extending to
plant and seed viability, and further to commercial crop productivity, and native
plant demographics.
From the limited number of studies reported to date, plants appear to have at
least some of the mechanisms for DNA repair found in other eukaryotic kingdoms.
Direct reversal pathways of DNA repair in plants have been demonstrated by the
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characterisation of photolyase genes specific for either cyclobutane pyrimidine
dimers, or 6-4 photoproducts. Evidence has been found for the presence of
nucleotide excision repair activity in both adult plants and germinating seeds.
This thesis describes the original isolation and characterisation of full-length
sequence homologues of both XPD and XPF, and partial sequences of ERCC1 and
XPG homologues from Arabidopsis. atXPD has also been shown to have an activity
contributing to the viability of a rad3 mutant. Structural analysis of the predicted
plant proteins indicated the putative presence of domains characteristic of
homologous NER proteins from other organisms in which the process of NER is
more fully understood. These observations constitute evidence that the plant
proteins also function in a NER system in vivo in Arabidopsis thaliana, so
supporting the existence of such a system. Nevertheless, alternative interpretations
are possible, such as that atXPF is present to code for a protein active only in a
recombinational pathway and not in an NER system, whilst atXPD codes for a
protein required only as a component of the transcriptional machinery, and again, is
not involved in NER. Firstly however, repair synthesis consistent with NER is
thought to occur in plants, as discussed in section 1.1.1. Secondly, Arabidopsis
mutants displaying a phenotype consistent with an NER mutation are known, for
example the uvh1-1 mutant (see Table 1.2). Finally, circumstantial genetic evidence
argues strongly against the alternative interpretation in that during the past four
years additional putative NER genes have been described from plant species. More
specifically, plant homologues of yeast and mammalian proteins thought to be
involved in the broader picture of NER are shown in Table 4.1. Considered in
conjunction, the four new plant NER homologues reported here and the putative
NER components from plants previously reported contribute to the conclusion that
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an NER system similar to those found in other organisms also exists in higher
plants. This system probably acts on lesions which either escape, or are not suitable
substrates for, photoreactivation. Coordination of the repair system with the plant
cell cycle, however, is apparently not directly homologous with mammalian
systems. In addition, the unique developmental program of plants and factors such
as polyploidy have unknown effects on the maintenance of DNA integrity in higher
plants.
In conclusion therefore, there is now convincing evidence at molecular
(homologous genes, prereplicative synthesis in germination) and phenotypic
(existence of discrete mutants apparently deficient in repair as evidenced by
UV-sensitivity) levels for a functional NER system in Arabidopsis thaliana and
probably higher plants in general. On a biochemical basis, this system probably has
many similarities to those found in other eukaryotes.
4.9 TLS systems in higher plants
The identification of putative RAD30 and REV3 homologues, as described in
sections 3.15.1 and 3.15.2, is the first molecular evidence for the existence of
translesion synthesis systems in plants. Furthermore, since these genes, and the
proteins they encode, have functions in separate TLS pathways, the presence of both
pathways is implied. Characterisation of the function of these hypothetical plant
pathways has yet to be undertaken, however on the basis of conservation of function
from other organisms, the consequences of an active plant TLS system may be
considered. Firstly, the potential for a TLS mediated mutation fixed in the genome
of the cell progeny to give rise to tumorigenic metastasis appears to be much
reduced, or abolished, in a plant system compared to an animal system. This is due
largely to the immobility of a plant cell, but is also enhanced by the senescence of
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large numbers of cells during differentiation, to form xylem tissue for example. The
apparent inability of a plant cell to embark upon uninduced growth and division
even when cell cycle control mechanisms are altered also contributes to this
stability. Secondly, if TLS can be a significant source of variation in evolution and
adaptation to changing environments, then organisms which produce large
quantities of gametes, such as the egg cells and pollen of many plants, would benefit
more than those organisms producing fewer gametes.
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5 Further work
The work presented in this thesis represents the first attempt of this
laboratory to define NER in plants by a PCR-based molecular approach, in contrast
to the genetic approach via plant mutants previously adopted. As such, it provides
the impetus for a range of projects, currently underway, to complete the
characterisation, and complementation assays, of the Arabidopsis atERCC1, atXPG,
atREV3 and atRAD30 homologues.
In addition, specific questions regarding the functions of atXPD and atXPF
remain unanswered. Firstly, the apparent inability of atXPD to completely replace
RAD3 in viability assays raises many intriguing questions about additional roles for
RAD3/XPD. Experiments to determine the growth rate and final titre at 39oC of the
D5225∆’32-38A strain carrying only RAD3 will shortly be instigated, to allow
resolution of the question of whether competition is really occurring between the
rad3ts14 protein and the wild-type RAD3. It is also intriguing to consider what might
result from viability complementation studies by atXPD with other rad3
temperature-sensitive mutants. Perhaps a more detailed “viability map” could be
derived. Modification of the atXPD cDNA to code for a histidine residue at the
codon equivalent to amino acid 661 of RAD3 may clarify the significance of this
particular variation.
Possession of the atXPD cDNA also allows the instigation of experiments to
discover, in a 2-hybrid system, which Arabidopsis proteins interact with atXPD,
perhaps including other TFIIH components (such as the plant homologue of p44),
but potentially also proteins involved in a cell cycle-NER linkage such as is seen in
mammals.
115
The failure of atXPF to complement the rad1 yeast mutant has tentatively
been attributed to lack of appropriate in vivo interactions with the yeast RAD10
protein. Antibody detection of the V5 epitope-tagged atXPF fusion protein from
total protein preparations will reveal whether translation of the mRNA detected in
the KAM1 yeast mutant actually occurs. The isolation and cloning of the atXPF and
atERCC1 cDNAs will shortly allow yeast mutant complementation assays of
combinations of atXPF, atERCC1, RAD1 and RAD10. In vitro interactions of these
four proteins (some as tagged fusion proteins) will also be assessed, using
non-denaturing polyacrylamide gel electrophoresis of buffered mixtures of the
purified proteins. Additionally, immunoaffinity column purification may be used to
withdraw these tagged proteins either individually or as complexes from yeast total
protein preparations.
RAD1 in S. cerevisiae is known to participate in mitotic recombination.
Assessment of the ability of atXPF to complement a RAD1 mutation for this
function is also currently underway in a collaborative project.
In conclusion, the results of this study contribute to the understanding of
mechanisms of DNA lesion repair in higher plants, by the original identification and
characterisation of putative NER genes and cDNAs. In addition, investigations
initiated by this study are currently underway to define several plant genes coding
for putative components of DNA lesion tolerance complexes. In total, this
information will allow a greater understanding of some of the fundamental
processes whereby higher plants, an economically and ecologically prominent group
of organisms, respond to challenges to their genomic integrity, particularly in an
increasingly hostile environment.
116
6 Recent Publications
Vonarx, E. J., Mitchell H., Karthikeyan R., Chatterjee, I., and Kunz, B. A.
(1998). DNA repair in higher plants. Mutation Research 400, 187-200.
Kunz, B. A., Karthikeyan, R., Vonarx, and E. J. (1998). DNA sequence
analysis of spontaneous mutagenesis in Saccharomyces cerevisiae. Genetics 148,
1491-1505.
Yang, Y., Karthikeyan, R., Mack, S. E., Vonarx, E. J., and Kunz, B. A.
(1999). Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in
mismatch correction efficiencies in prokaryotic and eukaryotic cells. Molecular and
General Genetics, 261 777-787.
Karthikeyan, R., Simon, M., Faye, G., Vonarx E. J., and Kunz B. A. (2000).
Evidence from mutational specificity studies that yeast DNA polymerases δ and ε
replicate different DNA strands at an intracellular replication fork. Journal of
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Kunz, B.A., Straffon, A.F.L., and Vonarx, E. J., (2000). DNA
damage-induced mutation: tolerance via translesion synthesis. Mutation Research
451 169-185.
Vonarx, E. J., Karthikeyan, R., Straffon, A., and Kunz, B. A. (2000). DNA
amplification and cycle sequencing of SUP4-o alleles directly from yeast colonies.
In preparation.
Vonarx, E. J., and Kunz, B. A. (2000). Variable transcript processing of
atXPF, an Arabidopsis homologue of the yeast RAD1 nucleotide excision repair
gene. In preparation.
Vonarx, E. J., and Kunz, B. A. (2000). An XPD homologue from
Arabidopsis thaliana has conserved domains and can functionally complement a
117
yeast RAD3 mutant for viability. Proceedings of the National Academy of Sciences
USA. In preparation.
Kunz, B. A., Suckling, N., Angelucci, C., Karthikeyan, R., and Vonarx, E. J.
(2000). Roles for the Saccharomyces cerevisiae REV3 polymerase ζ and the REV1
deoxycytidyl transferase in mutagenesis at endogenous abasic sites. In preparation.
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Figure 1.1 Schematic depiction of yeast NER proteins at a DNA lesion.
represents the lesion, for example a CPD.
Initial recognition and binding of the lesion is probably by the
RAD4/RAD23 complex, aided by, or subsequently stabilised by RAD14. RPA
also apparently has some function in stabilisation or formation of the assembling
NER complex. TFIIH is then recruited to the site, possibly via a direct interaction
between RAD4 and either the RAD25 or p62 TFIIH subunit. The helicase activity
of both RAD3 and RAD25 is required to create a local bubble of single-stranded
DNA (including the lesion). Incision of the single-stranded backbone either side
of the lesion is mediated by the RAD1/RAD10 endonucleolytic complex on the 5’
side, and RAD2 on the 3’ side, although RAD2 also has a structural role in the
complex. Completion of the incisions allows the removal of the single-stranded
segment incorporating the lesion, and its resynthesis using the complementary
strand as template.
Figure 2.1 atXPD 3’ RACE Strategy.
The 3’ untranslated region of atXPD was PCR-amplified and T/A cloned to
precisely locate the active stop codon and potentially elucidate details of the
polyadenylation processing of this gene. Primer sequences are shown in Table 2.1.
The 3’ RACE process is depicted as five separate steps. They are:
(a) synthesis of a cDNA molecule by a reverse transcriptase, using
poly T-priming of the mRNA template.
(b) digestion of the mRNA strand from the mRNA/cDNA hybrid
through the action of RNase H.
(c) annealing of the atXPD gene-specific primer AtmRNA-RAD3-SF5
to its complementary sequence on the cDNA, and synthesis of the second cDNA
strand.
(d) annealing of the adapter primer AP1 to the second cDNA strand,
and synthesis of the complementary strand by extension of the adapter primer.
(e) step (d) has resulted in generation of dsDNA flanked by the
gene-specific primer AtmRNA-RAD3-SF5 on one end, and the adapter-specific
primer Ent100-6 on the other.
(f) conventional PCR using the gene-specific nested primer
AtmRNA-RAD3-SF7, and Ent100-6 allows amplification of the cDNA region that
includes a short section of coding sequence, the STOP codon, the 3' untranslated
sequence and a portion of the poly A tail. This fragment can then be T/A cloned and
sequenced.
Figure 3.1 Multiple alignment of RAD3/XPD protein homologues.
Alignment of RAD3/XPD homologues from widely different phyla allowed
identification of regions common to all homologues, so allowing design of the
degenerate PCR primers RAD3-F1 and RAD3-R1, with which to amplify a genomic
DNA fragment from Arabidopsis.
Accession numbers of these entries are:
human XPD Accession Number 11950
Xiphophorus (fish) XPD Accession Number 2134009
S. pombe RAD15 Accession Number 1709995
S. cerevisiae RAD3 Accession Number 131812
This alignment was generated using the CLUSTALW software package, in
which * denotes identical amino acids at that position, and : or ⋅ denotes conservative
replacement.
human MKLNVDGLLVYFPYDYIYPEQFSYMRELKRTLDAKGHGVLEMPSGTGKTVSLLALIMAYQ Xiphophorus MKLNIEGLLVYFPYDYIYPEQYSYMLELKRTLDAKGHGVLEMPSGTGKTISLLSLIVAYQ S.pombe MKFYIDDLPILFPYPRIYPEQYQYMCDLKHSLDAGGIALLEMPSGTGKTISLLSLIVSYQ S.cerevisiae MKFYIDDLPVLFPYPKIYPEQYNYMCDIKKTLDVGGNSILEMPSGTGKTVSLLSLTIAYQ **: ::.* : *** *****:.** ::*::**. * .:**********:***:* ::** human RAYPLEVTKLIYCSRTVPEIEKVIEELRKLLNFYEKQEGEKLPFLGLALSSRKNLCIHPE Xiphophorus RAFPLDVTKLIYCSRTVPEIEKVVEELRKLMEFYAKETGEVNNFLALSLSSRKNLCIHPE S.pombe QHYP-EHRKLIYCSRTMSEIDKALAELKRLMAYRTSQLGYEEPFLGLGLTSRKNLCLHPS S.cerevisiae MHYP-EHRKIIYCSRTMSEIEKALVELENLMDYRTKELGYQEDFRGLGLTSRKNLCLHPE :* : *:******:.**:*.: **..*: : .: * * .*.*:******:**. human VTPLRFGKDVDGKCHSLTASYVRAQYQHDT--SLPHCRFYEEFDAHGREVPLPAGIYNLD Xiphophorus VSSLRFGKEVDGKCHSLTASYIRAQRHSNP--NQPVCRFYEEFDAVGRQVPLPAGIYNLD S.pombe VRREKNGNVVDARCRSLTAGFVREQRLAG--MDVPTCEFHDNLEDLEPHSLISNGVWTLD S.cerevisiae VSKERKGTVVDEKCRRMTNGQAKRKLEEDPEANVELCEYHENLYNIEVEDYLPKGVFSFE * : *. ** :*: :* . : : . . *.::::: . :. *::.:: human DLKALGRRQGWCPYFLARYSILHANVVVYSYHYLLDPKIADLVSKELARKAVVVFDEAHN Xiphophorus DLKDFGRRKGWCPYYLARYSILHANIVVYSYHYLLDPKIADLVSKELAKKSVVVFDEAHN S.pombe DITEYGEKTTRCPYFTVRRMLPFCNVIIYSYHYLLDPKIAERVSRELSKDCIVVFDEAHN S.cerevisiae KLLKYCEEKTLCPYFIVRRMISLCNIIIYSYHYLLDPKIAERVSNEVSKDSIVIFDEAHN .: .. ***: .* : .*:::************: **.*:::..:*:****** human IDNVCIDSMSVNLTRRTLDRCQGNLETLQKTVLRIKETDEQRLRDEYRRLVEGLREASAA Xiphophorus IDNVCIDSMSVNITRRTVDRCQNNVDTLQNTIHKIKETDAAKLREEYRRLVEGLKEANVA S.pombe IDNVCIESLSIDLTESSLRKASKSILSLEQKVNEVKQSDSKKLQDEYQKLVRGLQDANAA S.cerevisiae IDNVCIESLSLDLTTDALRRATRGANALDERISEVRKVDSQKLQDEYEKLVQGLHSADIL ******:*:*:::* :: :. . :*:: : .::: * :*::**.:**.**:.*. human RETDA-HLANPVLPDEVLQEAVPGSIRTAEHFLGFLRRLLEYVKWRLRVQHVVQESPPAF Xiphophorus RETDI-YLANPVLPDEVLQEAVPGSIRTAEHFVGFLRRFLEYLKARLRVQHVVQESAPQF S.pombe NDEDQ-FMANPVLPEDVLKEAVPGNIRRAEHFIAFLKRFVEYLKTRMKVLHVIAETPTSF S.cerevisiae TDQEEPFVETPVLPQDLLTEAIPGNIRRAEHFVSFLKRLIEYLKTRMKVLHVISETPKSF : : .: .****:::* **:**.** ****:.**:*::**:* *::* **: *:. *
human LSGLAQRVCIQRKPLRFCAERLRSLLHTLEITDLADFSPLTLLANFATLVSTYAKGFTII Xiphophorus LKDIFDKVCIDRKPLRFCAERLQCLLRTLEIADIADFSAVTLISNFATLVSTYSQGFTII S.pombe LQHVKDITFIDKKPLRFCAERLTSLVRALQISLVEDFHSLQQVVAFATLVATYERGFILI S.cerevisiae LQHLKQLTFIERKPLRFCSERLSLLVRTLEVTEVEDFTALKDIATFATLISTYEEGFLLI *. : : . *::******:*** *:::*::: : ** .: : ****::** .** :* human IEPFDDRTPTIANPILHFSCMDASLAIKPVFERFQSVIITSGTLSPLDIYPKILDFHPVT Xiphophorus IEPFEDRTPTIANPVLHFSCMDPSIAIKPVFQRFQSVIITSGTLSPLDIYPRILDFRPVT S.pombe LEPFETENATVPNPILRFSCLDASIAIKPVFERFRSVIITSGTLSPLDMYPKMLQFNTVM S.cerevisiae IEPYEIENAAVPNPIMRFTCLDASIAIKPVFERFSSVIITSGTISPLDMYPRMLNFKTVL :**:: ...::.**:::*:*:*.*:******:** ********:****:**::*:*..* human MATFTMTLARVCLCPMIIGRGNDQVAISSKFETREDIAVIRNYGNLLLEMSAVVPDGIVA Xiphophorus MASFTMTLARTCLCPLIVGRGNDQVALSSKFETREDFAVIRNYGNLLLEMSAVVPDGIVA S.pombe QESYGMSLARNCFLPMVVTRGSDQVAISSKFEARNDPSVVRNYGNILVEFSKITPDGLVA S.cerevisiae QKSYAMTLAKKSFLPMIITKGSDQVAISSRFEIRNDPSIVRNYGSMLVEFAKITPDGMVV :: *:**: .: *::: :*.****:**:** *:* :::****.:*:*:: :.***:*. human FFTSYQYMESTVASWYEQGILENIQRNKLLFIETQDGAETSVALEKYQEACENGRGAILL Xiphophorus FFTSYVYMENIVASWYEQGILENIQKNKLIFIETQDAAETSMALEKYQEACENGRGAILL S.pombe FFPSYLYLESIVSSWQSMGILDEVWKYKLILVETPDPHETTLALETYRAACSNGRGAVLL S.cerevisiae FFPSYLYMESIVSMWQTMGILDEVWKHKLILVETPDAQETSLALETYRKACSNGRGAILL **.** *:*. *: * ***::: : **:::** * **::***.*: **.*****:** human SVARGKVSEGIDFVHHYGRAVIMFGVPYVYTQSRILKARLEYLRDQFQIRENDFLTFDAM Xiphophorus SVARGKVSEGIDFVHHFGRAVIMFGVPYVYTQSRILKARLEYLRDQFQIRENDFLTFDAM S.pombe SVARGKVSEGVDFDHHYGRAVIMFGIPYQYTESRVLKARLEFLRDTYQIREADFLTFDAM S.cerevisiae SVARGKVSEGIDFDHQYGRTVLMIGIPFQYTESRILKARLEFMRENYRIRENDFLSFDAM **********:** *::**:*:*:*:*: **:**:******::*: ::*** ***:**** human RHAAQCVGRAIRGKTDYGLMVFADKRFARGDKRGKLPRWIQEHLTDANLNLTVDEGVQVA Xiphophorus RHAAQCLGRAIRGKTDYGLMIFADKRYARSDKRGKLPRWIQEHINDGSLNLTVDEAVQLS S.pombe RHAAQCLGRVLRGKDDHGIMVLADKRYGRSDKRTKLPKWIQQYITEGATNLSTDMSLALA S.cerevisiae RHAAQCLGRVLRGKDDYGVMVLADRRFSR--KRSQLPKWIAQGLSDADLNLSTDMAISNT ******:**.:*** *:*:*::**:*:.* ** :**:** : :.:. **:.* .: :
human KYFLRQMAQPFHREDQLGLSLLSLEQLESEETLKRIEQIAQQL------------------ Xiphophorus KHFLRQMAQPFRQEDQLGLSLLTLEQLQSEEMLQKISQMAHQA------------------ S.pombe KKFLRTMAQPFTASDQEGISWWSLDDLLIHQK-KALKSAAIEQSKHEDEMDIDVVET---- S.cerevisiae KQFLRTMAQPTDPKDQEGVSVWSYEDLIKHQNSRKDQGGFIENENKEGEQDEDEDEDIEMQ * *** **** .** *:* : ::* .: : . :
Figure 3.2 atXPD Genomic, cDNA and protein sequences.
The genomic sequence was derived from Genbank Accession number
AC005278. Exonic sequence is highlighted in bold type, and nucleotides are
numbered according to their position in the genomic sequence. Other features
highlighted are:
GeneTrapper capture oligonucleotide, RAD3-Cap
Primer AtmRNA-RAD3-SF7
Primer AtmRNA-RAD3-F1
Primer AtmRNA-RAD3-F2, including TAG stop codon
3’ untranslated sequence
Primer AP1
Putative consensus control sequence
Putative polyadenylation signal
M I F K I E D V T V Y F P Y D N I ATGATCTTTAAAATCGAAGACGTAACTGTCTACTTCCCTTATGACAACAT 0
Y P E Q Y E Y M V E L K R A L D ATATCCAGAACAATATGAATACATGGTTGAATTGAAGCGAGCCCTAGACG 50
A K G H C L L E M P T G T G K T I CTAAGGGACATTGTCTTCTCGAGATGCCTACCGGAACTGGTAAAACCATT 100
A L L S L I T S Y R L S R P D S P GCTCTTCTCTCTCTAATCACCAGCTATCGACTCTCTCGTCCTGATTCTCC 150
I K L V Y C T R T V H E M E K T GATCAAGCTTGTTTACTGTACTCGTACTGTCCACGAGATGGAGAAAACGT 200
L G E L K L L H D Y Q V R H L G T TAGGTGAGCTTAAGCTACTACATGACTACCAAGTACGCCATCTCGGCACC 250
Q A K I L A L G L S S R K N L C V CAGGCTAAGATCCTCGCCCTTGGTCTTTCCTCTCGGAAGAATCTTTGCGT 300
N T K V L A A E N R D S V D A A TAATACTAAGGTTTTAGCAGCCGAGAATCGTGATTCTGTTGATGCGGCCT 350
C R K R T A S W V R A L S T E N P GTAGGAAACGAACTGCTAGCTGGGTTAGGGCTTTGTCCACGGAGAATCCA 400
N V E L C D F F E N Y E K A A E N AATGTAGAACTCTGTGACTTCTTTGAGAACTATGAAAAGGCTGCTGAGAA 450
A L L P P G V Y T L E TGCGTTGTTGCCTCCTGGTGTTTATACTTTAGAGGTAAGATTAAAAAAGT 500
TAAGGTTTTTCACATTTGTTGTTTGATCAAGTTTATGTTTTAGTTGGAAT 550
GCTTATTTCGAGGTTACTTGCACATTTTGCCTGATGGTATATGTGTGTTT 600
D L R A F G K N R G W C P CGATTTTTAGGATCTAAGGGCGTTTGGTAAGAATCGTGGATGGTGCCCTT 650
Y F L A R H M I Q F A N V I V Y S ACTTTCTCGCGAGGCATATGATTCAGTTCGCCAACGTTATTGTTTACAGC 700
Y Q Y L L D P K V A G F I S K E L TACCAATACCTACTGGATCCTAAGGTTGCTGGGTTCATATCAAAGGAGTT 750
Q K E S V V V F D E A H N I D N ACAAAAAGAGTCAGTTGTAGTGTTTGACGAGGCCCATAATATCGATAACG 800
V C I E A L S V S V R R V T L E G TGTGTATTGAAGCACTCAGCGTCAGTGTGAGGAGGGTTACACTTGAAGGA 850
A N R N L N K I R Q E I D R GCTAATCGAAATCTTAATAAGATAAGGCAAGAAATTGATAGGTACGCTAA 900
TTTTTTCCTATATAGCTGATACTCCTTAAGTTCTCTTGAACGTGCCACTC 950
TTAATACAACAATAAACACAGTGATAGGGTCTTTTCTTTTTCAATTGAAT 1000
TTCCTGATTGTTCTTGAATGCCATCTGGGTGTCTGCTCACTCCAGTTGCT 1050
AATTGTTGGTTTTCTTAAGGAAGTTTAAGTTAGTAAGAAAATAATCTGGT 1100
TTTATGAGACTGCTTTTTGTCAAAGAACTGTAAGGGGAACAAAAATTTAC 1150
AAACGCTTTTGTCATCATCTTAAACACATGAAGCATAATTTTCTTGGTTC 1200
AATACAGGAAGTGACCTGGTTATTTGCTGATGCTTATTTTTAAGTTCTTA 1250
F K A T D TTACATTATTGAAATTGTCCAACTGGTAGGAAGGTTCAAGGCTACTGATG 1300
A G R L R A E Y N R L V E G L A L CAGGAAGATTGCGAGCTGAATACAACCGGCTGGTTGAGGGTTTAGCACTG 1350
R G D L S AGAGGGGACTTATCTGGTAAGTATGTGTTCAGGAAGGTGATCTGTGATTT 1400
GATTGAAAGCACACAGTAAATTTTCTAATTCTATGAACTTGACGCAACAA 1450
G G D Q W L A N P A L P TTTTTTTTTTGCAGGAGGTGATCAATGGCTTGCAAACCCTGCACTGCCTC 1500
H D I L K E A V P G N I R R A E H ATGATATTCTAAAGGAGGCTGTCCCGGGGAATATTAGACGGGCTGAGCAT 1550
F V H V L R R L L Q Y L G V R L D TTTGTGCATGTTTTACGCCGATTACTTCAGTACTTGGGGGTACGGCTGGA 1600
T E N V E K E S P V S F V S S L CACTGAGAATGTAGAGAAAGAAAGCCCTGTTAGCTTTGTATCGTCACTGA 1650
N S Q A G I E Q K T L K F C Y D R ATTCTCAGGCTGGAATTGAGCAGAAAACACTGAAGTTCTGTTATGACCGG 1700
L Q S L M L T L E I T D T D E F L CTCCAGTCTCTCATGCTAACCCTTGAAATTACAGATACAGATGAGTTTTT 1750
P I Q T V C D F A T L V G T Y A GCCTATCCAGACAGTGTGCGACTTTGCAACTCTTGTTGGGACATATGCCC 1800
R G F S I I I E P Y D E R M P H I GGGGATTTTCCATCATCATCGAGCCTTATGACGAGAGAATGCCCCATATT 1850
P D P I L Q CCAGATCCTATATTGCAGGTAAATATGGTGTTGACCTACTTTCTTTTGAG 1900
TATTGTCACTGGGATTTTAAGGGATTATGACTCACACGTAAATGCTACCG 1950
TTTGAATAATTGAGAATTGTATCTGAAACATATATCCTTTTGTATCTTAG 2000
L S C H D A S L A I K P V F D R F TTGAGCTGTCATGATGCGTCTCTGGCGATCAAACCGGTGTTTGATCGTTT 2050
Q S V V I T S G T L S P I D L Y CCAGTCAGTCGTGATTACATCAGGCACATTGAGCCCAATTGATCTTTACC 2100
P R L L N F T P V V S R S F K M S CCCGTCTTCTTAATTTCACTCCTGTTGTTAGTCGAAGCTTTAAGATGTCA 2200
M T R D C I C P M V L T R G S ATGACACGAGACTGCATCTGCCCTATGGTTCTAACTCGGGGAAGGTATGT 2250
GGTATATAAAAAGAATCCTGAACACTTAACTTATTTTTTAATTTTATATG 2300
D Q L P V S TCTTGACATTATTGGTTGTTTTCTTTTTCAGTGATCAGCTTCCTGTTAGC 2350
T K F D M R S D P G V V R N Y G K ACCAAATTTGACATGAGAAGTGATCCTGGTGTTGTGAGGAATTACGGAAA 2400
L L V E M V S I V P D G V V C F GCTTTTGGTAGAGATGGTATCTATTGTTCCTGATGGAGTTGTCTGCTTCT 2450
F V S Y S Y M D G I I A T W N E T TTGTTAGCTACTCATATATGGATGGCATTATTGCTACATGGAATGAAACT 2500
G I L E GGAATTCTGGAAGGTAAGGTCTTCTTCATCGCTTTACCATGCCAGTAATT 2550
TTTCAGAGTTTTGCATGAATTGGAATTTTTATGATACCTCAAAATATTTT 2600
TAGTTGCTTCAGGCATTAATGTATATTTGTTTACTGATATTACCAATTCT 2650
E I M Q Q K L V F I E T CTTAATGTTTGTAGGAAATAATGCAACAGAAGCTTGTTTTCATTGAAACA 2700
Q D V V E T T L A L D N Y R R A C CAAGATGTTGTAGAAACAACATTGGCACTGGATAATTATCGCAGGGCTTG 2750
D C G R G A V F F S V A R CGATTGCGGAAGAGGTGCAGTTTTCTTCTCTGTGGCCAGGTAAAATTTTT 2800
ATTTTTGAATGGTAGATGGTTTGGGAATTCATTGCAATGCGTCTTTGGTT 2850
CTGCTTCTTTGTTTCTGTATTATAATACTGGAATTGTCTCTTCTCTCCTA 2900
TTCTTGGTTGGTTAACAAATATTAAAGAGGTTTCACTTTTGAGAAATTTC 2950
G K V A E AAAGAAAGTGATCAAATTTTGTATTTGAACTGCAGGGGGAAAGTTGCTGA 3000
G I D F D R H Y G R L V V M Y G AGGTATCGATTTTGATCGTCATTATGGAAGACTGGTTGTAATGTACGGAG 3050
V P F Q Y T L S K TACCATTCCAGTATACATTAAGCAAGTAAGTTGTACTCAAATGAATGTTT 3100
ATCATTTCAATTTCCGGTTTTCTGCTTTTATATTTCATCGTACTTGGGAG 3150
I L R A R L ATGATAAGATGGAAACGGGAAAAATTTCAGGATATTACGAGCAAGATTGG 3200
E Y L H D T F Q I K E G D F L T F AGTATTTGCACGATACATTTCAAATAAAAGAAGGCGATTTCCTAACTTTT 3250
D A L GATGCCTTGGTATGATTCTTATTTTATGATCTTATTAGATTTTGGTGTGC 3300
AACTCTTTTACCTGTTTGTGACTGTTGTTAACTCTTTTTGGCTTTAACAC 3350
R Q A A Q C V G R V I R S K A D AGAGGCAAGCAGCTCAATGTGTAGGGCGAGTAATCCGGTCAAAGGCTGAT 3400
Y G M M I F A D K R TATGGGATGATGATATTTGCAGACAAAAGGTACATGCCCGCCTTAAGTTT 3450
TCTCTCTCTGAGTCTGGTCATTTAGATTCTTGCTATTTTACTAAATGGGA 3500
TTCTCTCTATAATCTATCTAGTATGATAATAAGTATATGCGCAAGTATCT 3550
GAGATCACCTCGAGAATAGAAGATTGTAACAGAGTTCACACTGTTCCTTA 3600
GGAATGGGAAAATATTCGTAGGACAGAAGAGAGACACAGATATATGCTTA 3650
TGTTGGTTTATAATGTCACATTTCTATGGTCTAACAATACACTGCAATTG 3700
Y S R H D K R S K L P ATGGTAAAACTTGCAGATACAGCCGTCATGATAAGCGGTCCAAACTACCT 3750
G W I L S H L R D A H L N L S T D GGTTGGATACTTTCGCATCTGCGTGATGCACACTTGAATCTAAGCACTGA 3800
M A I H I A R E TATGGCTATTCACATTGCTCGAGAGGTAAACCTATAACTCGCTGCTTTTT 3850
TATAACTCACAGTGAAACAAACAACAAGGCATAAAGAATCAACAATTTTC 3900
F L R K M A Q P Y CTGGTTTTTGTTTTTTTTGCAGTTTCTTCGGAAAATGGCTCAACCGTATG 3950
D K A G T M G R K T L L T Q E D L ATAAGGCGGGTACAATGGGAAGGAAAACTCTGTTGACACAAGAAGATTTG 4000
E K M A E T G V Q D M A Y STOP GAGAAGATGGCTGAGACTGGTGTGCAAGACATGGCTTATTAGTAAATCAG 4050 TTTGTTTGTTATTTTTTGGTTTAGGTCAATCAAAAATTGTTGTAGCAAAA 4100
ATTTTCACTGTAGGTATTTTGTTCTTTTTCTTGATGATTTTCAAGAATCG 4150
AATTGAAATTTAAGGCTCGTAAAAGTTTCGTTTTGGTTTTAAATCAAAAA 4200
AAAAAAAAAAAAAAATGGGCGCGGGGCATGACTAT 4250
Figure 3.3 atXPD protein alignment.
Alignment of RAD3/XPD homologues with the atXPD protein reveals
conservation of many amino acid residues. The locations of the seven conserved
helicase domains in RAD3 from S. cerevisiae are shown in italics (Deschavanne and
Harosh, 1993), and numbered I to VI above the corresponding locations. AtXPD
shows substantial sequence conservation in these, and other, regions of the
alignment.
Accession numbers of these entries are:
human XPD Accession Number 11950
Xiphophorus XPD Accession Number 2134009
Arabidopsis atXPD Accession Number AF188623 (this study)
S. pombe RAD15 Accession Number 1709995
S. cerevisiae RAD3 Accession Number 131812
This alignment was generated using the CLUSTALW software package, in which *
denotes identical amino acids at that position, and : or . denotes conservative
replacement.
I human MKLNVDGLLVYFPYDYIYPEQFSYMRELKRTLDAKGHGVLEMPSGTGKTVSLLALIMAYQ60 Xiphophorus MKLNIEGLLVYFPYDYIYPEQYSYMLELKRTLDAKGHGVLEMPSGTGKTISLLSLIVAYQ Arabidopsis MIFKIEDVTVYFPYDNIYPEQYEYMVELKRALDAKGHCLLEMPTGTGKTIALLSLITSYR60 S.pombe MKFYIDDLPILFPYPRIYPEQYQYMCDLKHSLDAGGIALLEMPSGTGKTISLLSLIVSYQ S.cerevisiae MKFYIDDLPVLFPYPKIYPEQYNYMCDIKKTLDVGGNSILEMPSGTGKTVSLLSLTIAYQ60 * : ::.: : *** *****:.** ::*::**. * :****:*****::**:* :*: II human RAYPLEVTKLIYCSRTVPEIEKVIEELRKLLNFYEKQEGEKLPFLGLALSSRKNLCIHPE120 Xiphophorus RAFPLDVTKLIYCSRTVPEIEKVVEELRKLMEFYAKETGEVNNFLALSLSSRKNLCIHPE Arabidopsis LSRPDSPIKLVYCTRTVHEMEKTLGELKLLHDYQVRHLGTQAKILALGLSSRKNLCVNTK120 S.pombe QHYP-EHRKLIYCSRTMSEIDKALAELKRLMAYRTSQLGYEEPFLGLGLTSRKNLCLHPS S.cerevisiae MHYP-EHRKIIYCSRTMSEIEKALVELENLMDYRTKELGYQEDFRGLGLTSRKNLCLHPE119 * . *::**:**: *::*.: **. * : . * : .*.*:******::.. human VTPLRFGKDVDGKCHSLTASYVRAQYQHDT--SLPHCRFYEEFDAHGREVPLPAGIYNLD178 Xiphophorus VSSLRFGKEVDGKCHSLTASYIRAQRHSNP--NQPVCRFYEEFDAVGRQVPLPAGIYNLD Arabidopsis VLAAENRDSVDAACRKRTASWVRALSTENP--NVELCDFFENYEKAAENALLPPGVYTLE178 S.pombe VRREKNGNVVDARCRSLTAGFVREQRLAG--MDVPTCEFHDNLEDLEPHSLISNGVWTLD S.cerevisiae VSKERKGTVVDEKCRRMTNGQAKRKLEEDPEANVELCEYHENLYNIEVEDYLPKGVFSFE179 * . ** *: * . : . . * :.:: . :. *::.:: III IV human DLKALGRRQGWCPYFLARYSILHANVVVYSYHYLLDPKIADLVSKELARKAVVVFDEAHN238 Xiphophorus DLKDFGRRKGWCPYYLARYSILHANIVVYSYHYLLDPKIADLVSKELAKKSVVVFDEAHN Arabidopsis DLRAFGKNRGWCPYFLARHMIQFANVIVYSYQYLLDPKVAGFISKELQKESVVVFDEAHN238 S.pombe DITEYGEKTTRCPYFTVRRMLPFCNVIIYSYHYLLDPKIAERVSRELSKDCIVVFDEAHN S.cerevisiae KLLKYCEEKTLCPYFIVRRMISLCNIIIYSYHYLLDPKIAERVSNEVSKDSIVIFDEAHN239 .: .. ***: .* : .*:::***:******:* :*.*: :..:*:****** V human IDNVCIDSMSVNLTRRTLDRCQGNLETLQKTVLRIKETDEQRLRDEYRRLVEGLREASAA298 Xiphophorus IDNVCIDSMSVNITRRTVDRCQNNVDTLQNTIHKIKETDAAKLREEYRRLVEGLKEANVA Arabidopsis IDNVCIEALSVSVRRVTLEGANRNLNKIRQEIDRFKATDAGRLRAEYNRLVEGLALRGDL298 S.pombe IDNVCIESLSIDLTESSLRKASKSILSLEQKVNEVKQSDSKKLQDEYQKLVRGLQDANAA S.cerevisiae IDNVCIESLSLDLTTDALRRATRGANALDERISEVRKVDSQKLQDEYEKLVQGLHSADIL299 ******:::*:.: :: . . : : : ..: * :*: **.:**.** .
VI human RETDA-HLANPVLPDEVLQEAVPGSIRTAEHFLGFLRRLLEYVKWRLRVQHVVQESPPAF357 Xiphophorus RETDI-YLANPVLPDEVLQEAVPGSIRTAEHFVGFLRRFLEYLKARLRVQHVVQESAPQF Arabidopsis SGGDQ-WLANPALPHDILKEAVPGNIRRAEHFVHVLRRLLQYLGVRLDTENVEKESPVSF357 S.pombe NDEDQ-FMANPVLPEDVLKEAVPGNIRRAEHFIAFLKRFVEYLKTRMKVLHVIAETPTSF S.cerevisiae TDQEEPFVETPVLPQDLLTEAIPGNIRRAEHFVSFLKRLIEYLKTRMKVLHVISETPKSF359 : : .*.**.::* **:**.** ****: .*:*:::*: *: . :* *:. * human LSGLAQRVCIQRKPLRFCAERLRSLLHTLEITDLADFSPLTLLANFATLVSTYAKGFTII417 Xiphophorus LKDIFDKVCIDRKPLRFCAERLQCLLRTLEIADIADFSAVTLISNFATLVSTYSQGFTII Arabidopsis VSSLNSQAGIEQKTLKFCYDRLQSLMLTLEITDTDEFLPIQTVCDFATLVGTYARGFSII417 S.pombe LQHVKDITFIDKKPLRFCAERLTSLVRALQISLVEDFHSLQQVVAFATLVATYERGFILI S.cerevisiae LQHLKQLTFIERKPLRFCSERLSLLVRTLEVTEVEDFTALKDIATFATLISTYEEGFLLI419 :. : . . *::*.*:** :** *: :*::: :* .: : ****:.** .** :* human IEPFDDRTPTIANPILHFSCMDASLAIKPVFERFQSVIITSGTLSPLDIYPKILDFHPVT477 Xiphophorus IEPFEDRTPTIANPVLHFSCMDPSIAIKPVFQRFQSVIITSGTLSPLDIYPRILDFRPVT Arabidopsis IEPYDERMPHIPDPILQLSCHDASLAIKPVFDRFQSVVITSGTLSPIDLYPRLLNFTPVV477 S.pombe LEPFETENATVPNPILRFSCLDASIAIKPVFERFRSVIITSGTLSPLDMYPKMLQFNTVM S.cerevisiae IEPYEIENAAVPNPIMRFTCLDASIAIKPVFERFSSVIITSGTISPLDMYPRMLNFKTVL479 :**:: . . :.:*:::::* *.*:******:** **:*****:**:*:**::*:* .* human MATFTMTLARVCLCPMIIGRGNDQVAISSKFETREDIAVIRNYGNLLLEMSAVVPDGIVA537 Xiphophorus MASFTMTLARTCLCPLIVGRGNDQVALSSKFETREDFAVIRNYGNLLLEMSAVVPDGIVA Arabidopsis SRSFKMSMTRDCICPMVLTRGSDQLPVSTKFDMRSDPGVVRNYGKLLVEMVSIVPDGVVC537 S.pombe QESYGMSLARNCFLPMVVTRGSDQVAISSKFEARNDPSVVRNYGNILVEFSKITPDGLVA S.cerevisiae QKSYAMTLAKKSFLPMIITKGSDQVAISSRFEIRNDPSIVRNYGSMLVEFAKITPDGMVV539 :: *:::: .: *::: :*.**:.:*::*: *.* .::****.:*:*: :.***:* VIIa VIIb human FFTSYQYMESTVASWYEQGILENIQRNKLLFIETQDGAETSVALEKYQEACENGRGAILL597 Xiphophorus FFTSYVYMENIVASWYEQGILENIQKNKLIFIETQDAAETSMALEKYQEACENGRGAILL Arabidopsis FFVSYSYMDGIIATWNETGILEEIMQQKLVFIETQDVVETTLALDNYRRACDCGRGAVFF597 S.pombe FFPSYLYLESIVSSWQSMGILDEVWKYKLILVETPDPHETTLALETYRAACSNGRGAVLL S.cerevisiae FFPSYLYMESIVSMWQTMGILDEVWKHKLILVETPDAQETSLALETYRKACSNGRGAILL599 ** ** *::. :: * ***::: : **:::** * **::**:.*: **. ****:::
human SVARGKVSEGIDFVHHYGRAVIMFGVPYVYTQSRILKARLEYLRDQFQIRENDFLTFDAM657 Xiphophorus SVARGKVSEGIDFVHHFGRAVIMFGVPYVYTQSRILKARLEYLRDQFQIRENDFLTFDAM Arabidopsis SVARGKVAEGIDFDRHYGRLVVMYGVPFQYTLSKILRARLEYLHDTFQIKEGDFLTFDAL657 S.pombe SVARGKVSEGVDFDHHYGRAVIMFGIPYQYTESRVLKARLEFLRDTYQIREADFLTFDAM S.cerevisiae SVARGKVSEGIDFDHQYGRTVLMIGIPFQYTESRILKARLEFMRENYRIRENDFLSFDAM659 *******:**:** :::** *:* *:*: ** *::*:****:::: ::*:* ***:***: human RHAAQCVGRAIRGKTDYGLMVFADKRFARGDKRGKLPRWIQEHLTDANLNLTVDEGVQVA717 Xiphophorus RHAAQCLGRAIRGKTDYGLMIFADKRYARSDKRGKLPRWIQEHINDGSLNLTVDEAVQLS Arabidopsis RQAAQCVGRVIRSKADYGMMIFADKRYSRHDKRSKLPGWILSHLRDAHLNLSTDMAIHIA717 S.pombe RHAAQCLGRVLRGKDDHGIMVLADKRYGRSDKRTKLPKWIQQYITEGATNLSTDMSLALA S.cerevisiae RHAAQCLGRVLRGKDDYGVMVLADRRFSR--KRSQLPKWIAQGLSDADLNLSTDMAISNT717 *:****:**.:*.* *:*:*::**:*:.* ** :** ** . : :. **:.* .: : human KYFLRQMAQPFHREDQLGLSLLSLEQLESEETLKRIEQIAQQL------------------ Xiphophorus KHFLRQMAQPFRQEDQLGLSLLTLEQLQSEEMLQKISQMAHQA------------------ Arabidopsis REFLRKMAQPYDKAGTMGRKTLLTQEDLEKMAETGVQDMAY-------------------- S.pombe KKFLRTMAQPFTASDQEGISWWSLDDLLIHQK-KALKSAAIEQSKHEDEMDIDVVET---- S.cerevisiae KQFLRTMAQPTDPKDQEGVSVWSYEDLIKHQNSRKDQGGFIENENKEGEQDEDEDEDIEMQ : *** **** . * . :: . .
Figure 3.4 UV-sensitivity complementation of the rad3-1 mutation.
Survival curves are shown for a wild-type strain, and an isogenic rad3-1
mutant strain (WS3-1) carrying the expression vector with RAD3, negative control
insert (atXPD in reverse orientation), or atXPD. The presence of the expression
vector carrying the RAD3 gene has restored survival of the rad3-1 mutant to the
wild-type level. In contrast, neither the negative control insert, nor the atXPD
insert altered the survival of the mutant.
Data points shown are the means of five repeats at each point.
Print from Excell
Figure 3.5 atXPD viability complementation assay in rad3ts14.
This photograph shows two 90 mm plates inoculated with 2,000 cells of
otherwise isogenic yeast strains carrying either rad3ts14 alone, rad3ts14 and RAD3,
or rad3ts14 and atXPD. One plate was incubated at 30oC, the other at 39oC, as
labelled, both for 5 days. The viability at 39oC of rad3ts14 was clearly much less
than that of rad3ts14 with atXPD, which also approximated that of rad3ts14 with
RAD3.
Figure 3.6 Growth curves of the rad3ts14, rad3ts14 RAD3, and rad3ts14
atXPD strains at 39oC.
Comparison of the growth rates and final titres of these three strains at 39oC
reveals a substantially faster growth rate of both the RAD3 and
atXPD-complemented strain compared to the strain reliant on the rad3ts14 allele
only.
Data points are the means of at least four repeats. The uncertainty in the titre
at each point was calculated as a percentage Standard Error, the largest of which was
2.3%, and so is too small to appear on the graph.
Figure 3.7 Growth curves of the rad3ts14, rad3ts14 RAD3, and rad3ts14
atXPD strains at 30oC.
Growth of these cultures at 30oC results in a final titre of each strain
substantially greater than the respective final titres seen at 39oC.
Data points are the means of at least four repeats. The uncertainty in the titre
at each point was calculated as a percentage Standard Error, the largest of which was
3.8%, and so is too small to be visible on the graph.
Figure 3.8 Multiple protein alignment of RAD1 homologues
A protein alignment of various RAD1/XPF homologues reveals conserved
regions of amino acid sequence, particularly in the C-terminal half of the proteins.
The amino acid region of the alignment corresponding to the atXPF cDNA
target sequence of the AtmRNA-RAD1-F1 amplification primer is highlighted in
colour.
Accession Numbers of these entries are:
mouse ERCC4 Accession Number 2896801
human XPF Accession Number Q92889
S. pombe RAD16 Accession Number P36617
S. cerevisiae RAD1 Accession Number P06777
This alignment was generated using the CLUSTALW software package, in
which a * denotes identical amino acids at that position, and : or . denotes
conservative replacement.
mouse ------------------------------------------------------------ human ------------------------------------------------------------ S.pombe ------------------------------------------------------------ S.cerevisiae MSQLFYQGDSDDELQEELTRQTTQASQSSKIKNEDEPDDSNHLNEVENEDSKVLDDDAVL mouse -----------------MEPGLSGERRSMAPLLEYERQQVL-ELLDSDGLVVCARGLGTD human ----------------------------MAPLLEYERQLVL-ELLDTDGLVVCARGLGAD S.pombe -----------------------METKVHLP-LAYQQQVFN-ELIEEDGLCVIAPGLSLL S.cerevisiae YPLIPNEPDDIETSKPNINDIRPVDIQLTLP-LPFQQKVVENSLITEDALIIMGKGLGLL * * :::: . .*: *.* : . **. mouse RLLYHFLRLHCHPA--------CLVLVLNTQPAE--------EEY--FINQLK------I human RLLYHFLQLHCHPA--------CLVLVLNTQPAE--------EEY--FINQLK------I S.pombe QIAANVLSYFAVPG--------SLLLLVGANVDD--------IEL--IQHEME------S S.cerevisiae DIVANLLHVLATPTSINGQLKRALVLVLNAKPIDNVRIKEALEELSWFSNTGKDDDDTAV : :.* . * .*:*::.:: : * : : : mouse EGVEHLPRRVTNEIASN-----SRYEVYTQGGIIFATSRILVVDFLTGRIPSDLITGILV human EGVEHLPRRVTNEITSN-----SRYEVYTQGGVIFATSRILVVDFLTDRIPSDLITGILV S.pombe HLEKKLITVNTETMSVD-----KREKSYLEGGIFAITSRILVMDLLTKIIPTEKITGIVL S.cerevisiae ESDDELFERPFNVVTADSLSIEKRRKLYISGGILSITSRILIVDLLSGIVHPNRVTGMLV . ..* : :: : .* : * .**:: *****::*:*: : .: :**::: mouse YRAHRIIESCQEAFILRLFRQKNKRGFIKAFTDNAVAFDTGFCHVERVMRNLFVRKLYLW human YRAHRIIESCQEAFILRLFRQKNKRGFIKAFTDNAVAFDTGFCHVERVMRNLFVRKLYLW S.pombe LHADRVVSTGTVAFIMRLYRETNKTGFIKAFSDDPEQFLMGINALSHCLRCLFLRHVFIY S.cerevisiae LNADSLRHNSNESFILEIYRSKNTWGFIKAFSEAPETFVMEFSPLRTKMKELRLKNVLLW .*. : . :**:.::*..*. ******:: . * : : :: * :::: ::
mouse PRFHVAVNSFLEQ----HKPEVVEIHVSMTPAMLAIQTAILDILNACLKELKCHNPS-LE human PRFHVAVNSFLEQ----HKPEVVEIHVSMTPTMLAIQTAILDILNACLKELKCHNPS-LE S.pombe PRFHVVVAESLEK----SPANVVELNVNLSDSQKTIQSCLLTCIESTMRELRRLNSAYLD S.cerevisiae PRFRVEVSSCLNATNKTSHNKVIEVKVSLTNSMSQIQFGLMECLKKCIAELSRKNPE-LA ***:* * . *: :*:*::*.:: : ** :: :: : ** *. * mouse VEDLSLENALGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQYDCVTFLN human VEDLSLENAIGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQYDCVTFLN S.pombe MEDWNIESALHRSFDVIVRRQLDSVWHRVSPKTKQLVGDLSTLKFLLSALVCYDCVSFLK S.cerevisiae LDWWNMENVLDINFIRSIDSVMVPNWHRISYESKQLVKDIRFLRHLLKMLVTSDAVDFFG :: .:*..: * : : . **::. ::*.** *: *: **. * *.* *: mouse LLESLRATEKVFG-----QNSGWLFLDASTSMFVNARARVYRVPDVKLNKKAKTSEKTSS human LLESLRATEKAFG-----QNSGWLFLDSSTSMFINARARVYHLPDAKMSKKEKISEKMEI S.pombe LLDTLVLSVNVSSYPSNAQPSPWLMLDAANKMIRVARDRVY---------KE--SE---G S.cerevisiae EIQ-LSLDANKPSVSRKYSESPWLLVDEAQLVISYAKKRIFY--------KN---EY--- :: * : . . * **::* : :: *: *:: * * mouse PEVQETKKELVLESNPKWEALTDVLKEIEAENK--ESEALGGPGRVLICASDDRTCCQLR human KEGEETKKELVLESNPKWEALTEVLKEIEAENK--ESEALGGPGQVLICASDDRTCSQLR S.pombe PNMDAIP---ILEEQPKWSVLQDVLNEVCHETMLADTDAETSNNSIMIMCADERTCLQLR S.cerevisiae ----------TLEENPKWEQLIHILHDISHERM--T-NHLQGP--TLVACSDNLTCLELA **.:***. * .:*::: * : . :: .:*: ** :* mouse DYLSAGAETFLLRLYRKTFEKDGKAEEVWVNVRKGDGPKRTTKSDKRPKAAPNKER--AS human DYITLGAEAFLLRLYRKTFEKDSKAEEVWMKFRKEDSSKRIRKSHKRPKDPQNKER--AS S.pombe DYLST----VTYD--NKDSLKNMNSKLVDY-FQWREQYRKMSKSIKKPE--PSKEREASN S.cerevisiae KVLNAS-----NK--KRGVRQVLLNKLKWY-RKQREETKKLVKEVQSQDTFPENATLNVS . :. .: : : : : :: *. : . .: .
mouse AKRGAPLKRKKQELTLTQVLGSAEEPPEDKALEEDLCRQTSSSPEGCGVEIKRES----- human TKERT-LKKKKRKLTLTQMVGKPEELEEEGDVEEGYRREISSSPESCPEEIKHEE----- S.pombe TTSRKGVPPSKRRRVRGGNNATSRTTSDNTDANDSFSRDLRLEKILLSHLSKR------- S.cerevisiae STFSKEQVTTKRRRTRGASQVAAVEKLRN--AGTNVDMEVVFEDHKLSEEIKKGSGDDLD :. .*:. . . : . : . *: mouse --FDLNVSSDA-AYGILKEPLTI-IHPLLGCSDP-------------------------- human --FDVNLSSDA-AFGILKEPLTI-IHPLLGCSDP-------------------------- S.pombe --YEPEVGND--AFEVIDDFNSIYIYSYNGERD--------------------------- S.cerevisiae DGQEENAANDSKIFEIQEQENEILIDDGDAEFDNGELEYVGDLPQHITTHFNKDLWAEHC : : ..* : : .: * * . * mouse ----YAL---------------TRVLHEVEPRYVVLYDAELTFVRQLEIYRASRPGKPLR human ----YAL---------------TRVLHEVEPRYVVLYDAELTFVRQLEIYRASRPGKPLR S.pombe ----------------------ELVLNNLRPRYVIMFDSDPNFIRRVEVYKATYPKRSLR S.cerevisiae NEYEYVDRQDEILISTFKSLNDNCSLQEMMPSYIIMFEPDISFIRQIEVYKAIVKDLQPK *::: * *:::::.: .*:*::*:*:* : mouse VYFLIYGGSTEEQRYLTALRKEKEAFEKLIREKASMVVP----EER-EGRDE-----TNL human VYFLIYGGSTEEQRYLTALRKEKEAFEKLIREKASMVVP----EER-EGRDE-----TNL S.pombe VYFMYYGGSIEEQKYLFSVRREKDSFSRLIKERSNMAIVLTADSERFESQESKFLRNVNT S.cerevisiae VYFMYYGESIEEQSHLTAIKREKDAFTKLIRENANLSHH----FETNEDLSH------YK ***: ** * *** :* ::::**::* :**:*.:.: * *. . mouse DLARGS--AALDAPTDTRKAGGQ--EQNGTQSSIVVDMREFRSELPSLIHRRGIDIEPVT human DLVRGT--ASADVSTDTRKAGGQ--EQNGTQQSIVVDMREFRSELPSLIHRRGIDIEPVT S.pombe RIAGGGQLSITNEKPRVRSLYLM--FICIKTLKVIVDLREFRSSLPSILHGNNFSVIPCQ S.cerevisiae NLAERKLKLSKLRKSNTRNAGGQQGFHNLTQDVVIVDTREFNASLPGLLYRYGIRVIPCM :. . .*. . ::** ***.:.**.::: .: : *
mouse LEVGDYILTPELCVERKSVSDLIGSLHSGRLYSQCLAMSRYYRRPVLLIEFDPSKPFSLA human LEVGDYILTPEMCVERKSISDLIGSLNNGRLYSQCISMSRYYKRPVLLIEFDPSKPFSLT S.pombe LLVGDYILSPKICVERKSIRDLIQSLSNGRLYSQCEAMTEYYEIPVLLIEFEQHQSFTSP S.cerevisiae LTVGDYVITPDICLERKSISDLIGSLQNNRLANQCKKMLKYYAYPTLLIEFDEGQSFSLE * ****:::*.:*:****: *** ** ..** .** * .** *.*****: :.*: mouse PRG--------------AFFQEMSSSDVSSKLTLLTLHFPRLRLLWCPSPHATAELFEEL human SRG--------------ALFQEISSNDISSKLTLLTLHFPRLRILWCPSPHATAELFEEL S.pombe PFS--------------DLSSEIGKNDVQSKLVLLTLSFPNLRIVWSSSAYVTSIIFQDL S.cerevisiae PFSERRNYKNKDISTVHPISSKLSQDEIQLKLAKLVLRFPTLKIIWSSSPLQTVNIILEL . . : .::...::. **. *.* ** *:::*..*. * :: :* mouse KQNKPQPDAATAMAITADSETLPESDRYNPGPQD--------FVLKMPGVNAKNCRSLMN human KQSKPQPDAATALAITADSETLPESEKYNPGPQD--------FLLKMPGVNAKNCRSLMH S.pombe KAMEQEPDPASAASIGLEAGQD-STNTYNQAPLD--------LLMGLPYITMKNYRNVFY S.cerevisiae KLGREQPDPSNAVILGTNKVRS-DFNSTAKGLKDGDNESKFKRLLNVPGVSKIDYFNLRK * . :**.:.* : : . : . * :: :* :. : .: mouse -QVKNIAELATLSLERLTTILGHSGNAKQLHDFLHTAYADLVSKGRVRK----------- human -HVKNIAELAALSQDELTSILGNAANAKQLYDFIHTSFAEVVSKGKGKK----------- S.pombe GGVKDIQEASETSERKWSELIGPEAG-RRLYSFFRKQLKDYE------------------ S.cerevisiae -KIKSFNKLQKLSWNEINELINDEDLTDRIYYFLRTEKEEQEQESTDENLESPGKTTDDN :*.: : * . . ::. ::: *::. : mouse -------------- human -------------- S.pombe -------------- S.cerevisiae ALHDHHNDVPEAPV
Figure 3.9 Multiple alignment of selected regions of UVH1 and atXPF
cDNA sequences.
Alignment of the nucleotide sequences of atXPF-1, -2, and -3 clones and the
UVH1 sequence (Liu et al., 2000) allows identification of the regions thought to
result from variable transcript processing (shown in colour), and also the A/G
transition at base number 1261 (in bold). START and STOP codons are shown in
bold. atXPF-3 and UVH1 are the most similar sequences of those aligned. Regions
of identical sequence across all four cDNAs have been omitted, and designated as
shown.
This alignment was generated using the CLUSTALW software package, in
which a * denotes identical amino acids at that position, and : or . denotes
conservative replacement.
UVH1 ATGGCGCTGAAATATCACCAACAGATCATA -1,230 intervening nucleotides- atXPF-2 ATGGCGCTGAAATATCACCAACAGATCATA -1,230 intervening nucleotides- atXPF-3 ATGGCGCTGAAATATCACCAACAGATCATA -1,230 intervening nucleotides- atXPF-1 ATGGCGCTGAAATATCACCAACAGATCATA -1,230 intervening nucleotides- ****************************** UVH1 GTGGCTTGCAAAGATGAACGGTCCTGCATGCAGCTTGAAGATTGCATCACAAACAATCCA atXPF-2 GTGGCTTGCAAAGATGAACGGTCCTGCATGCAGCTTGAAGGTTGCATCACAAACAATCCA atXPF-3 GTGGCTTGCAAAGATGAACGGTCCTGCATGCAGCTTGAAGGTTGCATCACAAACAATCCA atXPF-1 GTGGCTTGCAAAGATGAACGGTCCTGCATGCAGCTTGAAGGTTGCATCACAAACAATCCA **************************************** ******************* UVH1 CAGAAGGTAATGCGGGAAGAATGGGAAATG -690 intervening nucleotides- atXPF-2 CAGAAGGTAATGCGGGAAGAATGGGAAATG -690 intervening nucleotides- atXPF-3 CAGAAGGTAATGCGGGAAGAATGGGAAATG -690 intervening nucleotides- atXPF-1 CAGAAGGTAATGCGGGAAGAATGGGAAATG -690 intervening nucleotides- ****************************** UVH1 TCGATGATTATTCCCGTTGATCAG------------------------------------ atXPF-2 TCGATGATTATTCCCGTTGATCAGTTTCTTTTTCCTGCTTTCTTCTCATCCATACTGTGC atXPF-3 TCGATGATTATTCCCGTTGATCAG------------------------------------ atXPF-1 TCGATGATTATTCCCGTTGATCAG------------------------------------ ************************ UVH1 -----------------GATGGGCTCTGCATGGGGTCGAATTCTTCTACAGAGTTTCCAG atXPF-2 TATAAACTTGGAATTAGGATGGGCTCTGCATGGGGTCGAATTCTTCTACAGAGTTTCCAG atXPF-3 -----------------GATGGGCTCTGCATGGGGTCGAATTCTTCTACAGAGTTTCCAG atXPF-1 -----------------GATGGGCTCTGCATGGGGTCGAATTCTTCTACAGAGTTTCCAG ******************************************* UVH1 CTTCAAGTACACAAAACTCATTAACCCGAA -150 intervening nucleotides- atXPF-2 CTTCAAGTACACAAAACTCATTAACCCGAA -150 intervening nucleotides- atXPF-3 CTTCAAGTACACAAAACTCATTAACCCGAA -150 intervening nucleotides- atXPF-1 CTTCAAGTACACAAAACTCATTAACCCGAA -150 intervening nucleotides- ******************************
UVH1 CAATATGCGTAGAGAGAAAGAGTATTCAAGATCTTTTCCAGAGCTTCACATCAGGTCGTT atXPF-2 CAATATGCGTAGAGAGAAAGAGTATTCAAGATCTTTTCCAGAGCTTCACATCAGGTCGTT atXPF-3 CAATATGCGTAGAGAGAAAGAGTATTCAAGATCTTTTCCAGAGCTTCACATCAGGTCGTT atXPF-1 CAATATGCGTAGAGAGAAAGAGTATTCAAGATCTTTTCCAGAGCTTCACATCAG------ ****************************************************** UVH1 TATTTCACCAAGTCGAAATGATGTCCCGTTATTACAGAATACCAGTTCTTCTGATCGAGT atXPF-2 TATTTCACCAAGTCGAAATGATGTCCCGTTATTACAGAATACCAGTTCTTCTGATCGAGT atXPF-3 TATTTCACCAAGTCGAAATGATGTCCCGTTATTACAGAATACCAGTTCTTCTGATCGAGT atXPF-1 ------------------------------------------------------------ UVH1 TCTCACAAGACAAAAGCTTCTCCTTTCAGTCTTCGAGTGATATATCAGATGATGTGACTC atXPF-2 TCTCACAAGACAAAAGCTTCTCCTTTCAGTCTTCGAGTGATATATCAGATGATGTGACTC atXPF-3 TCTCACAAGACAAAAGCTTCTCCTTTCAGTCTTCGAGTGATATATCAGATGATGTGACTC atXPF-1 -----------------------------TCTTCGAGTGATATATCAGATGATGTGACTC ******************************* UVH1 CATATAACATCATATCAAAACTATCATTGC -330 intervening nucleotides- atXPF-2 CATATAACATCATATCAAAACTATCATTGC -330 intervening nucleotides- atXPF-3 CATATAACATCATATCAAAACTATCATTGC -330 intervening nucleotides- atXPF-1 CATATAACATCATATCAAAACTATCATTGC -330 intervening nucleotides- ****************************** UVH1 GTCTCAGAGAGTTTCTTGACGCCAAGTATCCGACTTTGCTATGA atXPF-2 GTCTCAGAGAGTTTCTTGACGCCAAGTATCCGACTTTGCTATGA atXPF-3 GTCTCAGAGAGTTTCTTGACGCCAAGTATCCGACTTTGCTATGA atXPF-1 GTCTCAGAGAGTTTCTTGACGCCAAGTATCCGACTTTGCTATGA ********************************************
Figure 3.10 atXPF Gene Structure
Comparison of the gene sequence and cDNA species identified in this
study, illustrating the variation in processing of exons 6 and 7 found in three
different atXPF cDNA species. Genomic base-pair number is shown along the top
of the gene sequence. Exons are depicted as open boxes and numbered in italics.
Intronic sequence is shown as a solid black line.
Print from powerpoint
RAD1 Gene structure.ppt
Figure 3.11 atXPF-3 cDNA and protein sequence
The atXPF cDNA is 2,871 nucleotides in length, and codes for a protein of
957 amino acids. The cDNA is presented here, with numbering below, and the
corresponding protein translation above.
Target sequences of primers AtmRNA-RAD1-F1 and R1 are highlighted in
red type. Primer At-mRNA-RAD1-F7 is shown in blue.
M A L K Y H Q Q I I S D L L E D S N G G
ATGGCGCTGAAATATCACCAACAGATCATATCAGACCTTCTCGAGGACTCTAATGGCGGA
1
L L I L S S G L S L A K L I A S L L I L
TTATTAATCCTTTCCTCCGGTCTCTCCCTCGCTAAATTAATCGCTTCTCTCCTCATCCTT
60
H S P S Q G T L L L L L S P A A Q S L K
CACTCTCCGTCGCAAGGAACTCTACTTCTCCTCCTCTCCCCCGCCGCTCAATCTCTAAAA
120
S R I I H Y I S S L D S P T P T E I T A
TCCAGAATCATCCATTACATCTCCTCTCTTGATTCTCCTACTCCGACGGAAATCACCGCA
180
D L P A N Q R Y S L Y T S G S P F F I T
GATCTTCCGGCGAATCAGCGTTACTCGCTTTACACCTCCGGTTCTCCTTTCTTCATCACG
240
P R I L I V D L L T Q R I P V S S L A G
CCGCGGATTCTCATCGTCGATCTGTTGACTCAACGGATTCCAGTTTCTTCACTCGCCGGA
300
I F I L N A H S I S E T S T E A F I I R
ATCTTCATCCTCAATGCTCATTCGATTTCCGAAACCTCCACTGAAGCATTCATTATACGA
360
I V K S L N S S A Y I R A F S D R P Q A
ATTGTCAAATCCCTAAATAGCTCTGCTTATATACGCGCTTTTTCCGATAGACCGCAAGCT
420
M V S G F A K T E R T M R A L F L R K I
ATGGTTTCCGGTTTCGCCAAGACTGAACGCACCATGCGTGCTCTCTTCCTCCGTAAAATC
480
H L W P R F Q L D V S Q E L E R E P P E
CATCTATGGCCAAGGTTTCAGTTGGATGTGTCTCAGGAATTGGAGCGAGAACCACCTGAG
540
V V D I R V S M S N Y M V G I Q K A I I
GTTGTGGATATTAGGGTTTCGATGTCGAATTACATGGTGGGAATACAGAAAGCTATCATT
600
E V M D A C L K E M K K T N K V D V D D
GAAGTAATGGATGCTTGTCTCAAGGAGATGAAGAAGACTAATAAAGTTGATGTTGATGAT
660
L T V E S G L F K S F D E I V R R Q L D
TTGACTGTAGAGAGTGGTTTGTTTAAGTCCTTTGATGAGATTGTGAGGAGGCAGCTTGAT
720
P I W H T L G K R T K Q L V S D L K T L
CCTATTTGGCATACTTTGGGGAAAAGGACAAAACAGCTTGTTTCTGATTTGAAGACTTTA
780
R K L L D Y L V R Y D A V S F L K F L D
AGGAAGTTGCTTGATTATCTTGTTAGGTATGATGCTGTGAGTTTTCTCAAGTTCTTGGAT
840
T L R V S E S Y R S V W L F A E S S Y K
ACACTAAGGGTATCAGAGAGCTATCGGTCTGTTTGGTTATTTGCAGAGTCCAGCTATAAA
900
I F D F A K K R V Y R L V K A S D V K S
ATATTTGATTTTGCAAAGAAACGAGTGTATCGTCTCGTGAAGGCAAGTGATGTCAAGTCA
960
K E H V K N K S G K K R N S K G E T D S
AAAGAACATGTTAAGAATAAGAGTGGTAAAAAGAGAAATTCGAAGGGCGAAACTGACTCT
1020
V E A V G G E T A T N V A T G V V V E E
GTAGAAGCAGTTGGTGGGGAAACGGCAACAAATGTGGCTACTGGCGTTGTTGTAGAAGAA
1080
V L E E A P K W K V L R E I L E E T Q E
GTTCTGGAAGAGGCACCAAAATGGAAAGTCTTACGTGAAATTTTAGAGGAGACACAAGAA
1120
E R L K Q A F S E E D N S D N N G I V L
GAAAGACTAAAGCAGGCTTTTTCTGAGGAAGATAACAGTGACAACAATGGAATAGTTCTT
1180
V A C K D E R S C M Q L E G C I T N N P
GTGGCTTGCAAAGATGAACGGTCCTGCATGCAGCTTGAAGGTTGCATCACAAACAATCCA
1240
Q K V M R E E W E M Y L L S K I E L R S
CAGAAGGTAATGCGGGAAGAATGGGAAATGTACCTGCTGAGCAAAATTGAGCTCCGCAGT
1300
M Q T P Q K K K Q K T P K G F G I L D G
ATGCAGACACCGCAAAAGAAGAAACAAAAGACTCCCAAGGGTTTTGGGATTCTTGATGGA
1360
V V P V T T I Q N S E G S S V G R Q E H
GTTGTTCCGGTAACTACAATACAGAACTCGGAAGGTAGCAGTGTTGGCAGACAAGAGCAT
1440
E A L M A A A S S I R K L G K T T D M A
GAGGCTCTTATGGCTGCTGCGTCTTCTATTCGCAAGCTAGGAAAGACAACAGATATGGCT
1500
S G N N N P E P H V D K A S C T K G K A
TCTGGAAATAACAACCCAGAGCCTCATGTTGATAAGGCTTCGTGTACCAAAGGAAAAGCT
1560
K K D P T S L R R S L R S C N K K T T N
AAAAAGGATCCAACAAGTCTCCGGAGATCCTTAAGAAGTTGCAATAAGAAAACGACGAAC
1620
S K P E I L P G P E N E E K A N E A S T
AGTAAACCTGAAATCTTACCAGGCCCAGAGAATGAAGAAAAAGCCAATGAAGCTAGCACG
1680
S A P Q E A N A V R P S G A K K L P P V
TCAGCCCCTCAAGAAGCCAATGCTGTTCGCCCCAGCGGTGCTAAAAAGCTACCGCCTGTG
1740
H F Y A L E S D Q P I L D I L K P S V I
CATTTTTATGCCTTAGAAAGCGATCAACCTATACTAGATATCTTGAAACCCTCTGTGATC
1800
I V Y H P D M G F V R E L E V Y K A E N
ATTGTCTACCATCCTGACATGGGTTTTGTCAGAGAACTTGAGGTTTACAAAGCAGAGAAC
1860
P L R K L K V Y F I F Y D E S T E V Q K
CCTCTCAGAAAGCTGAAAGTTTATTTTATTTTCTATGATGAGTCCACAGAAGTGCAGAAG
1920
F E A S I R R E N E A F E S L I R Q K S
TTTGAAGCAAGCATACGTCGAGAAAATGAAGCGTTTGAATCATTGATCAGGCAAAAGTCT
1980
S M I I P V D Q D G L C M G S N S S T E
TCGATGATTATTCCCGTTGATCAGGATGGGCTCTGCATGGGGTCGAATTCTTCTACAGAG
2040
F P A S S T Q N S L T R K A G G R K E L
TTTCCAGCTTCAAGTACACAAAACTCATTAACCCGAAAAGCAGGCGGAAGAAAGGAACTG
2100
E K E T Q V I V D M R E F M S S L P N V
GAGAAAGAGACACAGGTTATAGTTGACATGAGGGAGTTCATGAGCAGTCTACCAAATGTT
2160
L H Q K G M K I I P V T L E V G D Y I L
CTTCACCAGAAAGGCATGAAGATAATACCAGTTACACTAGAGGTCGGCGACTATATTCTA
2220
S P S I C V E R K S I Q D L F Q S F T S
TCTCCTTCAATATGCGTAGAGAGAAAGAGTATTCAAGATCTTTTCCAGAGCTTCACATCA
2280
G R L F H Q V E M M S R Y Y R I P V L L
GGTCGTTTATTTCACCAAGTCGAAATGATGTCCCGTTATTACAGAATACCAGTTCTTCTG
2340
I E F S Q D K S F S F Q S S S D I S D D
ATCGAGTTCTCACAAGACAAAAGCTTCTCCTTTCAGTCTTCGAGTGATATATCAGATGAT
2400
V T P Y N I I S K L S L L V L H F P R L
GTGACTCCATATAACATCATATCAAAACTATCATTGCTAGTTCTGCATTTTCCTCGGTTA
2460
R L L W S R S L H A T A E I F T T L K S
AGGTTATTGTGGTCTCGAAGTCTACATGCAACCGCAGAAATCTTTACTACCTTAAAATCC
2520
N Q D E P D E T R A I R V G V P S E E G
AACCAAGACGAGCCTGACGAGACTCGAGCAATAAGAGTTGGTGTGCCTTCAGAAGAAGGT
2580
I I E N D I R A E N Y N T S A V E F L R
ATCATAGAAAATGACATCAGAGCCGAGAACTATAACACATCTGCAGTTGAGTTTCTGAGA
2640
R L P G V S D A N Y R S I M E K C K S L
AGGCTTCCTGGAGTTTCGGATGCGAATTACAGATCGATTATGGAGAAATGTAAGAGTTTG
2700
A E L A S L P V E T L A E L M G G H K V
GCTGAGCTCGCGTCTTTGCCTGTTGAGACATTAGCAGAACTCATGGGTGGTCACAAAGTT
2760
A K S L R E F L D A K Y P T L L &
GCTAAAAGTCTCAGAGAGTTTCTTGACGCCAAGTATCCGACTTTGCTATGA
2820
Figure 3.12 Multiple sequence alignment of predicted atXPF and
UVH1 protein sequences.
Regions of the alignment in which there exists variation across the
alignment are highlighted in colour. In addition, the glycine/ aspartic acid amino
acid variation at position 417 (resulting from the G/A discrepancy between the
UVH1 and atXPF sequences at nucleotide position 1,251) is shown in bold.
This alignment was generated using the CLUSTALW software package, in
which a * denotes identical amino acids at that position, and : or . denotes
conservative replacement.
UVH1 MALKYHQQIISDLLEDSNGGLLILSSGLSLAKLIASLLILHSPSQGTLLLLLSPAAQSLK atXPF-3 MALKYHQQIISDLLEDSNGGLLILSSGLSLAKLIASLLILHSPSQGTLLLLLSPAAQSLK atXPF-1 MALKYHQQIISDLLEDSNGGLLILSSGLSLAKLIASLLILHSPSQGTLLLLLSPAAQSLK atXPF-2 MALKYHQQIISDLLEDSNGGLLILSSGLSLAKLIASLLILHSPSQGTLLLLLSPAAQSLK ************************************************************ UVH1 SRIIHYISSLDSPTPTEITADLPANQRYSLYTSGSPFFITPRILIVDLLTQRIPVSSLAG atXPF-3 SRIIHYISSLDSPTPTEITADLPANQRYSLYTSGSPFFITPRILIVDLLTQRIPVSSLAG atXPF-1 SRIIHYISSLDSPTPTEITADLPANQRYSLYTSGSPFFITPRILIVDLLTQRIPVSSLAG atXPF-2 SRIIHYISSLDSPTPTEITADLPANQRYSLYTSGSPFFITPRILIVDLLTQRIPVSSLAG ************************************************************ UVH1 IFILNAHSISETSTEAFIIRIVKSLNSSAYIRAFSDRPQAMVSGFAKTERTMRALFLRKI atXPF-3 IFILNAHSISETSTEAFIIRIVKSLNSSAYIRAFSDRPQAMVSGFAKTERTMRALFLRKI atXPF-1 IFILNAHSISETSTEAFIIRIVKSLNSSAYIRAFSDRPQAMVSGFAKTERTMRALFLRKI atXPF-2 IFILNAHSISETSTEAFIIRIVKSLNSSAYIRAFSDRPQAMVSGFAKTERTMRALFLRKI ************************************************************ UVH1 HLWPRFQLDVSQELEREPPEVVDIRVSMSNYMVGIQKAIIEVMDACLKEMKKTNKVDVDD atXPF-3 HLWPRFQLDVSQELEREPPEVVDIRVSMSNYMVGIQKAIIEVMDACLKEMKKTNKVDVDD atXPF-1 HLWPRFQLDVSQELEREPPEVVDIRVSMSNYMVGIQKAIIEVMDACLKEMKKTNKVDVDD atXPF-2 HLWPRFQLDVSQELEREPPEVVDIRVSMSNYMVGIQKAIIEVMDACLKEMKKTNKVDVDD ************************************************************ UVH1 LTVESGLFKSFDEIVRRQLDPIWHTLGKRTKQLVSDLKTLRKLLDYLVRYDAVSFLKFLD atXPF-3 LTVESGLFKSFDEIVRRQLDPIWHTLGKRTKQLVSDLKTLRKLLDYLVRYDAVSFLKFLD atXPF-1 LTVESGLFKSFDEIVRRQLDPIWHTLGKRTKQLVSDLKTLRKLLDYLVRYDAVSFLKFLD atXPF-2 LTVESGLFKSFDEIVRRQLDPIWHTLGKRTKQLVSDLKTLRKLLDYLVRYDAVSFLKFLD ************************************************************ UVH1 TLRVSESYRSVWLFAESSYKIFDFAKKRVYRLVKASDVKSKEHVKNKSGKKRNSKGETDS atXPF-3 TLRVSESYRSVWLFAESSYKIFDFAKKRVYRLVKASDVKSKEHVKNKSGKKRNSKGETDS atXPF-1 TLRVSESYRSVWLFAESSYKIFDFAKKRVYRLVKASDVKSKEHVKNKSGKKRNSKGETDS atXPF-2 TLRVSESYRSVWLFAESSYKIFDFAKKRVYRLVKASDVKSKEHVKNKSGKKRNSKGETDS ************************************************************
UVH1 VEAVGGETATNVATGVVVEEVLEEAPKWKVLREILEETQEERLKQAFSEEDNSDNNGIVL atXPF-3 VEAVGGETATNVATGVVVEEVLEEAPKWKVLREILEETQEERLKQAFSEEDNSDNNGIVL atXPF-1 VEAVGGETATNVATGVVVEEVLEEAPKWKVLREILEETQEERLKQAFSEEDNSDNNGIVL atXPF-2 VEAVGGETATNVATGVVVEEVLEEAPKWKVLREILEETQEERLKQAFSEEDNSDNNGIVL ************************************************************ UVH1 VACKDERSCMQLEDCITNNPQKVMREEWEMYLLSKIELRSMQTPQKKKQKTPKGFGILDG atXPF-3 VACKDERSCMQLEGCITNNPQKVMREEWEMYLLSKIELRSMQTPQKKKQKTPKGFGILDG atXPF-1 VACKDERSCMQLEGCITNNPQKVMREEWEMYLLSKIELRSMQTPQKKKQKTPKGFGILDG atXPF-2 VACKDERSCMQLEGCITNNPQKVMREEWEMYLLSKIELRSMQTPQKKKQKTPKGFGILDG *************.********************************************** UVH1 VVPVTTIQNSEGSSVGRQEHEALMAAASSIRKLGKTTDMASGNNNPEPHVDKASCTKGKA atXPF-3 VVPVTTIQNSEGSSVGRQEHEALMAAASSIRKLGKTTDMASGNNNPEPHVDKASCTKGKA atXPF-1 VVPVTTIQNSEGSSVGRQEHEALMAAASSIRKLGKTTDMASGNNNPEPHVDKASCTKGKA atXPF-2 VVPVTTIQNSEGSSVGRQEHEALMAAASSIRKLGKTTDMASGNNNPEPHVDKASCTKGKA ************************************************************ UVH1 KKDPTSLRRSLRSCNKKTTNSKPEILPGPENEEKANEASTSAPQEANAVRPSGAKKLPPV atXPF-3 KKDPTSLRRSLRSCNKKTTNSKPEILPGPENEEKANEASTSAPQEANAVRPSGAKKLPPV atXPF-1 KKDPTSLRRSLRSCNKKTTNSKPEILPGPENEEKANEASTSAPQEANAVRPSGAKKLPPV atXPF-2 KKDPTSLRRSLRSCNKKTTNSKPEILPGPENEEKANEASTSAPQEANAVRPSGAKKLPPV ************************************************************ UVH1 HFYALESDQPILDILKPSVIIVYHPDMGFVRELEVYKAENPLRKLKVYFIFYDESTEVQK atXPF-3 HFYALESDQPILDILKPSVIIVYHPDMGFVRELEVYKAENPLRKLKVYFIFYDESTEVQK atXPF-1 HFYALESDQPILDILKPSVIIVYHPDMGFVRELEVYKAENPLRKLKVYFIFYDESTEVQK atXPF-2 HFYALESDQPILDILKPSVIIVYHPDMGFVRELEVYKAENPLRKLKVYFIFYDESTEVQK ************************************************************ UVH1 FEASIRRENEAFESLIRQKSSMIIPVDQDGLCMGSNSSTEFPASSTQNSLTRKAGGRKEL atXPF-3 FEASIRRENEAFESLIRQKSSMIIPVDQDGLCMGSNSSTEFPASSTQNSLTRKAGGRKEL atXPF-1 FEASIRRENEAFESLIRQKSSMIIPVDQDGLCMGSNSSTEFPASSTQNSLTRKAGGRKEL atXPF-2 FEASIRRENEAFESLIRQKSSMIIPVDQFLFPAFFSSILCYKLGIRMGSAWGRILLQSFQ ****************************
UVH1 EKETQVIVDMREFMSSLPNVLHQKGMKIIPVTLEVGDYILSPSICVERKSIQDLFQSFTS atXPF-3 EKETQVIVDMREFMSSLPNVLHQKGMKIIPVTLEVGDYILSPSICVERKSIQDLFQSFTS atXPF-1 EKETQVIVDMREFMSSLPNVLHQKGMKIIPVTLEVGDYILSPSICVERKSIQDLFQSFTS atXPF-2 LQVHKTH----------------------------------------------------- UVH1 GRLFHQVEMMSRYYRIPVLLIEFSQDKSFSFQSSSDISDDVTPYNIISKLSLLVLHFPRL atXPF-3 GRLFHQVEMMSRYYRIPVLLIEFSQDKSFSFQSSSDISDDVTPYNIISKLSLLVLHFPRL atXPF-1 VFE--------------------------------------------------------- atXPF-2 ------------------------------------------------------------ UVH1 RLLWSRSLHATAEIFTTLKSNQDEPDETRAIRVGVPSEEGIIENDIRAENYNTSAVEFLR atXPF-3 RLLWSRSLHATAEIFTTLKSNQDEPDETRAIRVGVPSEEGIIENDIRAENYNTSAVEFLR atXPF-1 ------------------------------------------------------------ atXPF-2 ------------------------------------------------------------ UVH1 RLPGVSDANYRSIMEKCKSLAELASLPVETLAELMGGHKVAKSLREFLDAKYPTLL atXPF-3 RLPGVSDANYRSIMEKCKSLAELASLPVETLAELMGGHKVAKSLREFLDAKYPTLL atXPF-1 -------------------------------------------------------- atXPF-2 --------------------------------------------------------
Figure 3.13 Multiple protein alignment of RAD1 and RAD1
homologues including atXPF-3.
Functional features of RAD1 are marked, including the putative
RAD10-binding site (Bardwell et al., 1993), and possible nuclear localisation
sequence with the associated casein kinase II phosphorylation site (Rihs et al.,
1991; Friedberg et al., 1995). The 14 amino acid sequence referred to in section
4.3 is further highlighted within the RAD10-binding region in blue type
Accession Numbers of the alignment members are:
mouse ERCC4 Accession Number 2896801
human XPF Accession Number 2842712
D. melanogaster MEI-9 Accession Number 3914026
N. crassa MUS38 Accession Number 3219304
S. cerevisiae RAD1 Accession Number 131810
atXPF-3 This study Accession Number AF191494
This alignment was generated using the CLUSTALW software package, in which
* denotes identical amino acids at that position, and : or . denotes conservative
replacement.
mouse ------------------------------------------------------------ human ------------------------------------------------------------ D.melanogaster ------------------------------------------------------------ atXPF-3 ------------------------------------------------------------ N.crassa ------------------------------------------------------------ S.cerevisiae MSQLFYQGDSDDELQEELTRQTTQASQSSKIKNEDEPDDSNHLNEVENEDSKVLDDDAVL mouse ----------------MEPGLSGERRSMAPLLEYERQQVLELLD-SDG-LVVCARGLGTD human ---------------------------MAPLLEYERQLVLELLD-TDG-LVVCARGLGAD D.melanogaster ----------------------------MVLLDYEKQMFLDLVE-ADG-LLVCAKGLSYD atXPF-3 -----------------------------MALKYHQQIISDLLEDSNGGLLILSSGLSLA N.crassa ------------------------------------------------------------ S.cerevisiae YPLIPNEPDDIETSKPNINDIRPVDIQLTLPLPFQQKVVENSLITEDA-LIIMGKGLGLL mouse RLLYHFLRLHCHP--------ACLVLVLN---------TQPAEEEYFINQL-------KI human RLLYHFLQLHCHP--------ACLVLVLN---------TQPAEEEYFINQL-------KI D.melanogaster RVVISILKAYSDS--------GNLVLVIN---------SSDWEEQYYKS---------KI atXPF-3 KLIASLLILHSPSQ-------GTLLLLLSPA-------AQSLKSRIIHYIS-------SL N.crassa -----------------------MPGLVR---------PWPRHAAISMSP--------KA S.cerevisiae DIVANLLHVLATPTSINGQLKRALVLVLNAKPIDNVRIKEALEELSWFSNTGKDDDDTAV : :: . mouse EGV----EHLPRRVTNEIASNS-RYEVYTQGGIIFATSRILVVDFLTGRIPSDLITGILV human EGV----EHLPRRVTNEITSNS-RYEVYTQGGVIFATSRILVVDFLTDRIPSDLITGILV D.melanogaster E---------PKYVHEGASTATERERVYLEGGLQFISTRILVVDLLKQRIPIELISGIIV atXPF-3 DS------PTPTEITADLPANQ-RYSLYTSGSPFFITPRILIVDLLTQRIPVSSLAGIFI N.crassa RG--------LTVVNTDFTSVGTREKMYAQGGIFSITSRILVVDLLTNLLNPETITGMLV S.cerevisiae ESDDELFERPFNVVTADSLSIEKRRKLYISGGILSITSRILIVDLLSGIVHPNRVTGMLV : : * :* .*. :.***:**:*. : . ::*:::
mouse YRAHRIIESCQEAFILRLFRQKNKRGFIKAFTDNAVAFDTGFCHVERVMRNLFVRKLYLW human YRAHRIIESCQEAFILRLFRQKNKRGFIKAFTDNAVAFDTGFCHVERVMRNLFVRKLYLW D.melanogaster LRAHTIIESCQEAFALRLFRQKNKTGFVKAFSSSPEAFTIGYSHVERTMRNLFVKHLYIW atXPF-3 LNAHSISETSTEAFIIRIVKSLNSSAYIRAFSDRPQAMVSGFAKTERTMRALFLRKIHLW N.crassa LHADRIVATSLEAFIFRIYRQKNKVGFLKAFSDNPDPFTVGFSPLATMMRNMFLRKASLW S.cerevisiae LNADSLRHNSNESFILEIYRSKNTWGFIKAFSEAPETFVMEFSPLRTKMKELRLKNVLLW .*. : .. *:* :.: :. *. .:::**:. . .: :. *: : ::: :* mouse PRFHVAVNSFLE-QHKP---EVVEIHVSMTPAMLAIQTAILDILNACLKELKCHNPSLEV human PRFHVAVNSFLE-QHKP---EVVEIHVSMTPTMLAIQTAILDILNACLKELKCHNPSLEV D.melanogaster PRFHESVRTVLQ-PWKI---QSIEMHVPISQNITSIQSHILEIMNFLVQEIKRINRTVDM atXPF-3 PRFQLDVSQELE-REPP---EVVDIRVSMSNYMVGIQKAIIEVMDACLKEMKKTN-KVDV N.crassa PRFHVQVAQSLEGKKKA---EVIELEVPMTDSMREIQTAIMECVEISIHELKKENTGLEM S.cerevisiae PRFRVEVSSCLNATNKTSHNKVIEVKVSLTNSMSQIQFGLMECLKKCIAELSRKNPELAL ***: * *: : :::.*.:: : ** ::: :. : *:. * : : mouse EDLSLENALGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQYDCVTFLNL human EDLSLENAIGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQYDCVTFLNL D.melanogaster EAVTVENCVTKTFHKILQAQLDCIWHQLNSQTKLIVADLKILRTLMISTMYHDAVSAYAF atXPF-3 DDLTVESGLFKSFDEIVRRQLDPIWHTLGKRTKQLVSDLKTLRKLLDYLVRYDAVSFLKF N.crassa EDWNLDSALTRNFDRMVRRQLEPNWHRVSWKTKQIAGDLTVLRGMLQSLLALDAVSFLQQ S.cerevisiae DWWNMENVLDINFIRSIDSVMVPNWHRISYESKQLVKDIRFLRHLLKMLVTSDAVDFFGE : .::. : * . : : ** :. .:* :. *: ** :: *.* mouse LE-SLRATEKVFGQ-----NSGWLFLDASTSMFVNARARVYRVPDVKLNK-----KAKTS human LE-SLRATEKAFGQ-----NSGWLFLDSSTSMFINARARVYHLPDAKMSK-----KEKIS D.melanogaster MK-RYRSTEYALS------NSGWTLLDAAEQIFKLSRQRVF------N------------ atXPF-3 LD-TLRVSESYRS--------VWLFAESSYKIFDFAKKRVYRLVKASDVKSKEHVKNKSG N.crassa LD-TIHAAHKPAPGTTRQTESPWLFSDAAQTIFETARQRVYS------SK------QKAG S.cerevisiae IQLSLDANKPSVSRKYS--ESPWLLVDEAQLVISYAKKRIFY------------------ :. . * : : : :: :: *::
mouse EKTSSPE---------VQETK-------KELVLESNPKWEALTDVL-KEIEAENKESE-A human EKMEIKE---------GEETK-------KELVLESNPKWEALTEVL-KEIEAENKESE-A D.melanogaster ----------------GQ----------QEFEPEPCPKWQTLTDLLTKEIPGDMRRSRRS atXPF-3 KKRNSKGETDSVEAVGGETATNVATGVVVEEVLEEAPKWKVLREILEETQEERLKQAFSE N.crassa P--NS----------TIES---------LKPVLEEQPKWAVLADVL-EEIDRDLYFEPAV S.cerevisiae -----------------KN----------EYTLEENPKWEQLIHIL-HDISHERMTNH-- : : * *** * .:* . mouse LGGPG---RVLICASDDRTCCQLRDYLSAG--AETFLLRLYR---KTFEKDGKAEEVWVN human LGGPG---QVLICASDDRTCSQLRDYITLG--AEAFLLRLYR---KTFEKDSKAEEVWMK D.melanogaster EQQP----KVLILCQDARTCHQLKQYLTQG--GPRFLLQQAL---QHEVPVGKLSDNYAK atXPF-3 EDNSDNNGIVLVACKDERSCMQLEGCITNN--PQKVMREEWE---MYLLSKIELRSMQTP N.crassa RDDSN--GTILVMCADTDTCRQLRDYLQTMHIRPRTAKKVEE---VYDPEEDRPSGAFLM S.cerevisiae LQGP-----TLVACSDNLTCLELAKVLNASN-KKRGVRQVLLNKLKWYRKQREETKKLVK . *: . * :* :* : . . mouse -VRKGDGPK--------------RTTKSDKRPKAAPN--KERASAKRGAPLK-------- human -FRKEDSSK--------------RIRKSHKRPKDPQN--KERASTKERT-LK-------- D.melanogaster ESQTRSAPP--------------KNVSSNKELR------REEVSGSQPP-LA-------- atXPF-3 QKKKQKTPKGFGILDGVVPV---TTIQNSEGSSVGRQ--EHEALMAAASSIR-------- N.crassa RRKLRNYLNWKREFAQVNAT---LFSENQKALSGAVDPRLPQARGRGGAPAN-------- S.cerevisiae EVQSQDTFPENATLNVSSTFSKEQVTTKRRRTRGASQVAAVEKLRNAGTNVDMEVVFEDH : . . . . . mouse RKKQELTLTQVLGSAEEPPEDKALEEDLCR----QTSSSPEG---CGVEIKRE---SFDL human KKKRKLTLTQMVGKPEELEEEGDVEEGYRR----EISSSPES---CPEEIKHE---EFDV D.melanogaster GMD-E--LAQLLSESE--TEGQHFEESYML----TMTQPVEVG-PAAIDIKPDPDVSIFE atXPF-3 KLGKTTDMASGNNNPEPHVDKASCTKGKAKKDPTSLRRSLRSCNKKTTNSKPEILPGPEN N.crassa KRRRVRGGGGGVGSNPSRHENGSIVQHFEKP---NEVADLMS---EIQITEDDAGGQAAE S.cerevisiae KLSEEIKKGSGDDLDDGQEENAANDSKIFEIQEQENEILIDDGDAEFDNGELEYVGDLPQ . : . : :
mouse NVSSDAAYGILKEPLTIIHP----LLGCSDPYALTR--------VLHEVEPRYVVLYDAE human NLSSDAAFGILKEPLTIIHP----LLGCSDPYALTR--------VLHEVEPRYVVLYDAE D.melanogaster TIPELEQFDVTAALASVPHQ----PYICLQTFKTEREGSMALEHMLEQLQPHYVVMYNMN atXPF-3 EEKANEASTSAPQEANAVRPSGAKKLPPVHFYALESD-----QPILDILKPSVIIVYHPD N.crassa EIITFATADPLEDMDDYYQLYDMQDLVVIHAYEGDQD-----EHVLEEVKPKYIIMYEPD S.cerevisiae HITTHFNKDLWAEHCNEYEYVDRQDEILISTFKSLND-----NCSLQEMMPSYIIMFEPD . : *. : * ::::. : mouse LTFVRQLEIYRASR---PGKPLRVYFLIYGGSTEEQRYLTALRKEKEAFEKLIREKASMV human LTFVRQLEIYRASR---PGKPLRVYFLIYGGSTEEQRYLTALRKEKEAFEKLIREKASMV D.melanogaster VTPIRQLEVFEARRRLPPADRMKVYFLIHARTVEEQAYLTSLRREKAAFEFIIDTKSKMV atXPF-3 MGFVRELEVYKAEN---PLRKLKVYFIFYDESTEVQKFEASIRRENEAFESLIRQKSSMI N.crassa ASFIRRVEVYRSSHN---DRNVRVYFLYYGGSVEEQRYLSSVRREKDAFTKLIRERASMS S.cerevisiae ISFIRQIEVYKAIVK---DLQPKVYFMYYGESIEEQSHLTAIKREKDAFTKLIRENANLS :*.:*::.: :***: : : * * . :::::*: ** :* .:.: mouse VPEEREGR-DETNLDLARG-SAALDAPTDTRKAG-GQE--QNGTQSS--IVVDMREFRSE human VPEEREGR-DETNLDLVRG-TASADVSTDTRKAG-GQE--QNGTQQS--IVVDMREFRSE D.melanogaster IPKYQDGKTDEAFLLLKTYDDEPTDENAKSRQAG-GQA--PQATKETPKVIVDMREFRSD atXPF-3 IPVDQDGLCMGSNSSTEFP--ASSTQNSLTRKAG-GR---KELEKET-QVIVDMREFMSS N.crassa IVMTTDSH------GVEDP-ESAFLRTINTRIAGGGKL--AKATAQPPRVVVDVREFRSS S.cerevisiae HHFETNEDLSHYKNLAERKLKLSKLRKSNTRNAG-GQQGFHNLTQDV--VIVDTREFNAS : . :* ** *: : . ::** *** :. mouse LPSLIHRRGIDIEPVTLEVGDYILTPELCVERKSVSDLIGSLHSGRLYSQCLAMSRYYRR human LPSLIHRRGIDIEPVTLEVGDYILTPEMCVERKSISDLIGSLNNGRLYSQCISMSRYYKR D.melanogaster LPCLIHKRGLEVLPLTITIGDYILTPDICVERKSISDLIGSLNSGRLYNQCVQMQRHYAK atXPF-3 LPNVLHQKGMKIIPVTLEVGDYILSPSICVERKSIQDLFQSFTSGRLFHQVEMMSRYYRI N.crassa LPSLLHGRSMVIVPCMLTVGDYILSPNICVERKSVSDLISSFKDGRLYAQCETMFQHYRN S.cerevisiae LPGLLYRYGIRVIPCMLTVGDYVITPDICLERKSISDLIGSLQNNRLANQCKKMLKYYAY ** ::: .: : * : :***:::*.:*:****:.**: *: ..** * * ::*
mouse PVLLIEFDPSKPFSLAPRGA-------------FFQEMSSS-DVSSKLTLLTLHFPRLRL human PVLLIEFDPSKPFSLTSRGA-------------LFQEISSN-DISSKLTLLTLHFPRLRI D.melanogaster PILLIEFDQNKPFHLQGKFM-------------LSQQTSMANDIVQKLQLLTLHFPKLRL atXPF-3 PVLLIEFSQDKSFSFQSSSD-------------ISDDVTPY-NIISKLSLLVLHFPRLRL N.crassa PMLLIEFDQNKSFTLEPFAD-----LSGSLRSVNPENAGAQRLAVQKSCLLTLAFPKLRI S.cerevisiae PTLLIEFDEGQSFSLEPFSERRNYKNKDISTVHPISSKLSQDEIQLKLAKLVLRFPTLKI * *****. .:.* : * *.* ** *:: mouse LWCPSPHATAELFEELKQNKPQPDAATAMAITA-----DSETLPE-------SDRYNPGP human LWCPSPHATAELFEELKQSKPQPDAATALAITA-----DSETLPE-------SEKYNPGP D.melanogaster IWSPSPYATAQLFEELKLGKPEPDPQTAAALGS-----DEPMAGE-------QLHFNSGI atXPF-3 LWSRSLHATAEIFTTLKSNQDEPDETRAIRVGVP----SEEGIIEND---IRAENYNTSA N.crassa IWSSSPYETAEIFERLKALEEEPDPVAAVRAGLGEGESPEDGVNEGKGAVNGGSTFNMEA S.cerevisiae IWSSSPLQTVNIILELKLGREQPDPSNAVILGTNKVRSDFNSTAKG-----LKDGDNESK :*. * *.::: ** . :** * : * mouse QDFVLKMPGVNAKNCRSLMNQVKNIAELATLSLERLTTILGHSGNAKQLHDFLHTAYADL human QDFLLKMPGVNAKNCRSLMHHVKNIAELAALSQDELTSILGNAANAKQLYDFIHTSFAEV D.melanogaster YDFLLRLPGVHTRNIHGLLRKGGSLRQLLLRSQKELEELLQSQESAKLLYDILHVAHLPE atXPF-3 VEFLRRLPGVSDANYRSIMEKCKSLAELASLPVETLAELMGGHKVAKSLREFLDAKYPTL N.crassa QEMLGKVPGVTPKNIRNITAEAENVREVANMEVEELGRLVG-REAAGKIVEFFKKDVLED S.cerevisiae FKRLLNVPGVSKIDYFNLRKKIKSFNKLQKLSWNEINELINDEDLTDRIYYFLRTEKEEQ . : .:*** : .: . .. :: . : :: : : :: mouse VSKGRVRK---------------------------------------------------- human VSKGKGKK---------------------------------------------------- D.melanogaster KDEVTGSTALLAA--SKQFGAGSHNRFRMAARIAKGSPMMLSSKDGP------------- atXPF-3 L----------------------------------------------------------- N.crassa YSG--------------------------------------------------------- S.cerevisiae EQESTDENLESPGKTTDDNALHDHHNDVPEAPV
Figure 3.14 UV-C sensitivity of yeast wild-type and rad1 mutants.
The isogenic wild-type, or KAM1 yeast strain (rad1 mutant) carrying a multicopy
plasmid expressing: 1. yeast wild-type RAD1, 2. atXPF-3, or 3. a negative control
insert, were exposed to controlled doses of UV-C radiation. The sensitivity of
each strain is indicated by the fraction of cells surviving after exposure to
increasing incident UV-C doses. Data points are the means of five repeats at each
exposure.
UV-sensitivity of recombinant yeast rad1 strains
0.00001
0.0001
0.001
0.01
0.1
1
10
100
0 5 10
UV-C dose (J/m2)
% S
urvi
val wild-type/atXPF
wild-typerad1/RAD1rad1/atXPFrad1/-ve
Figure 3.15 Multiple protein alignment of RAD10 homologues.
Alignment of RAD10/ERCC1 homologues reveals conservation of
specific regions across the alignment, particularly at the carboxyl terminus ends
of the proteins. The XPF-binding region of human ERCC1 is highlighted in red,
while the 12 amino acid C-terminal sequence within this region and known to be
required for resistance to UV-radiation or mitomycin-C is highlighted in blue.
The putative XPA-binding region is shown in pink type.
The Accession Numbers of the alignment entries are:
human ERCC1 Accession No. P07992
mouse ERCC1 Accession No. P07903
L. longifolium ERCC1 Accession No. CAA05781.1
atERCC1 Accession No. AF276082 (This study), and derived
from genomic clone of Accession No. AAF27027.1
S. pombe SWI10 Accession No. Q06182
S. cerevisiae RAD10 Accession No. P06838
human --------------MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVAKPLFRSTQSL mouse --------------MDPGKDEESRPQPSGPPTRRKFVIPLEEEEVPCAGVKPLFRSSRNP L.longifolium ------------------------------------------------------------ atERCC1 MANEDDDGEKSRSLHQQIARKPKTQIVIGVPSYQEVLESSQTKSTPPSLFKPSQSFSQAF S.pombe ------------------------------------------------------------ S.cerevisiae ------------------------MNNTDPTSFESILAGVAKLRKEKSGADTTG--SQSL human PTVDTS-AQAAPQTYAEYAISQPLEGAGATCPTGSE---PLAGETPNQ-ALKPG------ mouse TIPATS-AHMAPQTYAEYAITQPPGGAGATVPTGSE---PAAGENPSQ-TLKTG------ L.longifolium -----------PKP----AHESPP-------PQSSS---SSTGAAAP--AVAAG------ atERCC1 AFVKSSDVYSPPPPSSAAASSSQPSGA-SQVPHSSSQTHQTDGASSSSTPVATGSVPSNT S.pombe ------------------MSDIDDEEFEQLAVSALEEVEKKAGFAQQPTPQKVSR----- S.cerevisiae EIDASKLQQQEPQTSRRINSNQVINAFNQQKPEEWTDSKATDDYNRKR-PFRST------ . . human -AKSNSIIVSPRQRGNPVLKFVRNVPWEFG------DVIPDYVLGQSTCALFLSLRYHNL mouse -AKSNSIIVSPRQRGNPVLKFVRNVPWEFG------EVIPDYVLGQSTCALFLSLRYHNL L.longifolium -QNRNAILVSHRQKGNPLLKHIRNVKWVFA------DIVCDYLVGQSSCALYLSLRYHLL atERCC1 TQNRNAILVSHRQKGNPLLKHIRNVKWVFS------DIIPDYVLGQNSCALYLSLRYHLL S.pombe -VTAHSILVNPRQKGNPLLPHVRNVPWEYT------DIVPDFVMGTGICSLFLSLKYHHL S.cerevisiae -RPGKTVLVNTTQKENPLLNHLKSTNWRYVSSTGINMIYYDYLVR-GRSVLFLTLTYHKL ::::*. *: **:* .::.. * : : *::: . . *:*:* ** * human HPDYIHGRLQSLGKNFALRVLLVQVDVKDPQQALKELAKMCILADCTLILAWSPEEAGRY mouse HPDYIHERLQSLGKNFALRVLLVQVDVKDPQQALKELAKMCILADCTLVLAWSAEEAGRY L.longifolium HPDYLYYRIRELQKNFKLRVVLCHVDVEDVVKPLLEVTRTAMLHDCTLLCGWSLEECGRY atERCC1 HPDYLYFRIRELQKNFKLSVVLCHVDVEDTVKPLLEVTKTALLHDCTLLCAWSMTECARY S.pombe HPEYIYSRISKLGKSYNLRILLILVDVENHQASIQELVKTSIVNQYTLILAWSSEEAARY S.cerevisiae YVDYISRRMQPLSRNENN-ILIFIVDDNNSEDTLNDITKLCMFNGFTLLLAFNFEQAAKY : :*: *: * :. ::: ** :: .: ::.: .:. **: .:. :..:*
human LETYKAYEQKPADLLMEKLEQDFVSRVTECLTTVKSVNKTDSQTLLTTFGSLEQLIAASR mouse LETYRAYEQKPADLLMEKLEQNFLSRATECLTTVKSVNKTDSQTLLATFGSLEQLFTASR L.longifolium LETIKVYENKPADSIREQMDTDYLSRLTQALTSVRHVNKTDAVTLGTAFGSLSGIMDASL atERCC1 LETIKVYENKPADLIQGQMDTDYLSRLNHSLTSIRHVNKSDVVTLGSTFGSLAHIIDASM S.pombe LETYKAYENMSPALIMEKPSTDYLSQVQSFLTSIRGINKSDSLSLLSKFGSLERALVASR S.cerevisiae IEYLNL------------------------------------------------------ :* . human EDLALCPGLGPQKARRLFDVLHEPFLKVP------------------------------- mouse EDLALCPGLGPQKARRLFEVLHEPFLKVPR------------------------------ L.longifolium EDLARCPGIGERKVKRLHDTFHEPFRRVSN--RPAVAYLKLPPKMLSAEEILATKEGVEV atERCC1 EDLARCPGIGERKVKRLYDTFHEPFKRATSS-YPSVVEPPIP--EAPVEKDVNSEEPVEE S.pombe DELEQLEGWGPTKVNRFLEAVQQPFMSHSTIKRPEAINLKQT------------------ S.cerevisiae ------------------------------------------------------------ human ------------------------------------------------------------ mouse ------------------------------------------------------------ L.longifolium EG---EKANTSKCKESG------RALGQL------------------------------- atERCC1 DEDFVEDSRKRKKKEPEPEKTVKTALSAVFARYSDRLSKKKEKQKEKDTTTASDAETHQN S.pombe ------------------------------------------------------------ S.cerevisiae ------------------------------------------------------------
Figure 3.16 atXPG partial cDNA and protein sequence.
PCR amplification, cloning and sequencing of a fragment of the atXPG
cDNA has resulted in the nucleotide and protein sequences displayed. This
sequence corresponds to amino acids 789 to 1044 of the human XPG protein.
Features highlighted include: Primer AtmRNA-RAD2-F2
Primer AtmRNA-RAD2-R1
E A E A Q C A F M E Q S N L V D G I V T GGAAGCAGAGGCACAGTGTGCATTTATGGAACAATCAAACCTTGTTGATGGCATTGTCAC D D S D V F L F G A R S V Y K N I F D D TGACGATTCTGATGTATTCTTGTTTGGAGCAAGGAGCGTGTACAAAAATATATTTGATGA R K Y V E T Y F M K D I E K E L G L S R TCGTAAATATGTGGAGACATACTTTATGAAGGACATTGAGAAGGAGTTAGGCTTGAGCAG D K I I R M A M L L G S D Y T E G I S G AGATAAAATAATCCGCATGGCAATGCTTCTAGGAAGTGATTACACAGAAGGAATCAGTGG I G I V N A I E V V T A F P E E D G L Q TATTGGTATTGTCAATGCTATTGAAGTTGTGACTGCTTTTCCCGAGGAGGATGGGCTTCA K F R E W V E S P D P T I L G K T D A K GAAATTTCGTGAATGGGTTGAATCACCTGATCCAACCATTTTAGGAAAGACTGATGCAAA T G S K V K K R G S A S V D N K G I I S AACAGGTTCAAAGGTAAAGAAGCGTGGGTCTGCCTCCGTAGATAATAAAGGAATCATCTC G A S T D D T E E I K Q I F M D Q H R K AGGTGCCTCGACTGATGACACCGAAGAAATCAAGCAAATCTTCATGGATCAGCATAGAAA V S K N W H I P L T F P S E A V I S A Y GGTAAGCAAGAACTGGCATATTCCATTGACTTTCCCTAGTGAGGCTGTTATATCAGCCTA L N P Q V D L S T E K F S W G K P D L S CTTAAATCCGCAAGTGGATCTGTCGACTGAGAAATTCTCATGGGGCAAACCTGATCTTTC V L R K L C W E K F N W N G K K T D E L AGTACTTAGAAAGCTATGCTGGGAAAAGTTTAATTGGAACGGCAAGAAAACAGATGAACT L L P V L K E Y E K R E T Q L R I E A F ACTACTACCAGTCTTGAAGGAGTATGAAAAGCGCGAGACGCAACTTCGGATTGAAGCTTT Y S F N E R F A K I R S K R I N K A V K CTATTCCTTTAATGAAAGATTTGCAAAGATCCGTAGTAAAAGAATCAACAAAGCTGTCAA G I G G G L S S D V A D H T L Q E G P R AGGAATTGGTGGAGGTCTGTCGTCAGATGTGGCCGATCATACTCTCCAGGAAGGTCCAAG K R N AAAACGAAATAA
Figure 3.17 Multiple alignment of partial RAD2/XPG homologues.
Alignment of the regions of RAD2/XPG homologues corresponding to the
partial atXPG cDNA sequence sequenced thus far reveals substantial conservation
of residues throughout the alignment. Amino acids 789 to 1044 of the human
protein are shown in this alignment. The glutamine residue at position 791 of the
human sequence known to be essential for wild-type NER incision activity is
highlighted in red type. The putative XPG/PCNA interaction domain is
highlighted in blue type.
Accession numbers of these entries are:
mouse XPG Accession Number 549454
human XPG Accession Number 267420
Xenopus XPG Accession Number 267421
S. pombe RAD13 Accession Number 131777
S. cerevisiae RAD2 Accession Number 131811
This alignment was generated using the CLUSTALW software package, in which
* denotes identical amino acids at that position, and : or . denotes conservative
replacement.
mouse IQAPMEAEAQCAVLDLSDQTSGTITDDSDIWLFGARHVYKNFFNKNKFVEYYQYVDFYSQ human IQAPMEAEAQCAILDLTDQTSGTITDDSDIWLFGARHVYRNFFNKNKFVEYYQYVDFHNQ Xenopus IVAPMEAEAQCAILDLTDQTSGTITDDSDIWLFGARHVYKNFFSQNKHVEYYQYADIHNQ atXPG -----EAEAQCAFMEQSNLVDGIVTDDSDVFLFGARSVYKNIFDDRKYVETYFMKDIEKE S.pombe IVAPQEAEAQCSKLLELKLVDGIVTDDSDVFLFGGTRVYRNMFNQNKFVELYLMDDMKRE S.cerevisiae ITAPMEAEAQCAELLQLNLVDGIITDDSDVFLFGGTKIYKNMFHEKNYVEFYDAESILKL ******: : . ..* :*****::***. :*:*:* ..:.** * .: mouse LGLDRNKLINLAYLLGSDYTEGIPTVGCVTAMEILNEFPGRGLDPLLKFSEWWHEAQ--- human LGLDRNKLINLAYLLGSDYTEGIPTVGCVTAMEILNEFPGHGLEPLLKFSEWWHEAQ--- Xenopus LGLDRSKLINLAYLLGSDYTEGIPTVGYVSAMEILNEFPGQGLEPLVKFKEWWSEAQ--- atXPG LGLSRDKIIRMAMLLGSDYTEGISGIGIVNAIEVVTAFPEE--DGLQKFREWVESPDPTI S.pombe FNVNQMDLIKLAHLLGSDYTMGLSRVGPVLALEILHEFPGD--TGLFEFKKWFQRLS--- S.cerevisiae LGLDRKNMIELAQLLGSDYTNGLKGMGPVSSIEVIAEF-GN----LKNFKDWYNNGQ--- :.:.: .:*.:* ******* *: :* * ::*:: * * :* .* . mouse ----------NNKKVAENPYD---------------------TKVKKKLRK--LQLTPGF human ----------KNPKIRPNPHD---------------------TKVKKKLRT--LQLTPGF Xenopus ----------KDKKMRPNPND---------------------TKVKKKLRL--LDLQQSF atXPG LGKTDAKTGSKVKKRGSASVDNKGIISGASTDDTEEIKQIFMDQHRKVSKN--WHIPLTF S.pombe -------TGHASKNDVNTPVK---------------------KRINKLVGK--IILPSEF S.cerevisiae --------FDKRKQETENKFE---------------------KDLRKKLVNNEIILDDDF : . .* : * mouse PNPAVADAYLRPVVDDSRGSFLWGKPDVDKIREFCQRYFGWNRMKTDESLYPVLKHLNAH human PNPAVAEAYLKPVVDDSKGSFLWGKPDLDKIREFCQRYFGWNRTKTDESLFPVLKQLDAQ Xenopus PNPAVASAYLKPVVDESKSAFSWGRPDLEQIREFCESRFGWYRLKTDEVLLPVLKQLNAQ atXPG PSEAVISAYLNPQVDLSTEKFSWGKPDLSVLRKLCWEKFNWNGKKTDELLLPVLKEYEKR S.pombe PNPLVDEAYLHPAVDDSKQSFQWGIPDLDELRQFLMATVGWSKQRTNEVLLPVIQDMHKK S.cerevisiae PSVMVYDAYMRPEVDHDTTPFVWGVPDLDMLRSFMKTQLGWPHEKSDEILIPLIRDVNKR *. * .**:.* ** . * ** **:. :*.: ..* :::* * *:::. . :
mouse Q---TQLRIDSFFRLAQQEKQD---AKLIKSHRLSRAVTCMLRKEREEK--APELTKVTE human Q---TQLRIDSFFRLAQQEKED---AKRIKSQRLNRAVTCMLRKEKEAA--ASEIEAVSV Xenopus Q---TQLRIDSFFRLEQHE------AAGLKSQRLRRAVTCMKRKERDVE--AEEVEAAVA atXPG E---TQLRIEAFYSFNERF------AKIR-SKRINKAVKGIGGGLSSDV--ADHTLQEGP S.pombe QFVGTQSNLTQFFEGGNTNVYAPRVAYHFKSKRLENALSSFKNQISNQSPMSEEIQADAD S.cerevisiae KKKGKQKRINEFFPREYISGD----KKLNTSKRISTATGKLKKRKM-------------- : .* .: *: *:*: * : mouse AM-EKEFELLDDAKGKTQKR-ELPYKKETSVPKRRRPSGN------GGFLGDPYCSESPQ human AM-EKEFELLDKAKRKTQKRGITNTLEESSSLKRKRLSDSKRKNTCGGFLGETCLSESSD Xenopus VM-ERECTNQRKGQKTNTKSQGTKRRKPTECSQEDQDPGG-------GFIGIELKTLSSK atXPG RK-RNK------------------------------------------------------ S.pombe AFGESKGS--DELQSRILRR-----KKMMAS-KNSSDSDS-------DSEDNFLASLTPK S.cerevisiae ------------------------------------------------------------
Figure 3.18 Multiple protein alignment of RAD30 homologues.
The alignment of RAD30 homologues from both humans, and Saccharomyces,
with the putative atRAD30 protein allows an assessment of the degree of amino
acid conservation between these proteins. As shown, some regions of the
alignment show complete conservation, whereas other areas are more variable. If
only the human protein and atRAD30 are considered, substantially greater
similarity is found.
The Accession Numbers of these entries are:
atRAD30 2660675
human 5138988
S. cerevisiae 2131479
This alignment was generated using the CLUSTALW software package, in which
* denotes identical amino acids at that position, and : or . denotes conservative
replacement.
atRAD30 ---------MPVARPE---ASDARVIAHVDMDCFYVQVEQRKQPELRGLPSAVVQYNEWQ human ---------MATGQ--------DRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWK S.cerevisiae MSKFTWKELIQLGSPSKAYESSLACIAHIDMNAFFAQVEQMRCGLSKEDPVVCVQWN--- : . :* :**:.*:.**** : : * . **:: atRAD30 GGGLIAVSYEARKCGVKRSMRGDEAKAACPQIQLVQVPVAR-GKADLNLYRSAGSEVDG- human GGGIIAVSYEARAFGVTRSMWADDAKKLCPDLLLAQVRESR-GKANLTKYREASVEV--- S.cerevisiae --SIIAVSYAARKYGISRMDTIQEALKKCSNLIPIHTAVFKKGEDFWQYHDGCGSWVQDP .:***** ** *:.* ::* *.:: :. : *: : .. * atRAD30 --SGSYYYTVCVVSILAKSGK------------CERASIDEVYLDLTDAAESMLADAP-- human ------------MEIMSRFAV------------IERASIDEAYVDLTSAVQERLQKLQGQ S.cerevisiae AKQISVEDHKVSLEPYRRESRKALKIFKSACDLVERASIDEVFLDLGRICFNMLMFDNEY :. : . *******.::** . * atRAD30 PESLELIDEEVLKSHILGMN------REDGDDFKESVR------NWICREDAD----RRD human PISADLLPSTYIEGLPQGPT------TAEETVQKEGMRKQGLF-QWLDSLQIDNLT-SPD S.cerevisiae ELTGDLKLKDALSNIREAFIGGNYDINSHLPLIPEKIKSLKFEGDVFNPEGRDLITDWDD : :* . :.. . . * :: : : * * atRAD30 KLLSCGIIIVAELRKQVLKETEFTCSAGIAHNKMLAKLASGMNKPAQQTVVPYAAVQELL human LQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNRQTLVSHGSVPQLF S.cerevisiae VILALGSQVCKGIRDSIKDILGYTTSCGLSSTKNVCKLASNYKKPDAQTIVKNDCLLDFL *: * : :* : : *.*:: .* :.***.. :** **:* .: ::: atRAD30 SS--LPIKKMKQLGGKLGTSLQTDLGV--------------DTVGDLLQFSETKL-QEHY human SQ--MPIRKIRSLGGKLGASVIEILGI--------------EYMGELTQFTESQL-QSHF S.cerevisiae DCGKFEITSFWTLGGVLGKELIDVLDLPHENSIKHIRETWPDNAGQLKEFLDAKVKQSDY . : * .: *** ** .: *.: : *:* :* :::: *..: atRAD30 GVNTG-----------TWLWNIARGISGEEVQGRLLPKSHGSGKTFPGPRALKSLSTVQH human GEKNG-----------SWLYAMCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQW S.cerevisiae DRSTSNIDPLKTADLAEKLFKLSRGRYGLPLSSRPVVKSMMSNKNLRGK-SCNSIVDCIS . ... *: :.** :. * : *: ..*.: * : :
atRAD30 WLNQLSEELSERLGSDLEQNKRIASTLTLHASAFRVPTSLSKDSDSHKKFPSKSCPM-RY human WLLQLAQELEERLTKDRNDNDRVATQLVVSIRVQ---------GDKRLSSLRRCCALTRY S.cerevisiae WLEVFCAELTSRIQDLEQEYNKIVIPRTVSISLK----------TKSYEVYRKSGPVAYK ** :. ** .*: . :: .::. .: . . :. .: atRAD30 GVTKIQEDAFNLFQAALREYMGSFGIKPQGNKLETWRITGLSVSASKIVDIPSGTSSIMR human DAHKMSHDAFTVIKNCNTSGIQTEWSPP----LTMLFLCATKFSASAPSSSTDITSFLSS S.cerevisiae GINFQSHELLKVGIKFVTDLDIKGKNKS------YYPLTKLSMTITN-FDIIDLQKTVVD . ..: :.: . . . : ..: : . . . : atRAD30 YFQSQPTVPSRSADGCVQGN-----VAMTASASEGCSEQRSTETQ-------------AA human DPSSLPKVPVTSSEAKTQGSGPAVTATKKATTSLESFFQKAAERQKVKEASLSSLTAPTQ S.cerevisiae MFGNQVHTFKSSAGKEDEEKTTSSKADEKTPKLECCKYQVTFTDQK----------ALQE . . *: : . . .:. . * : * atRAD30 MPEVDTGVTYTLPNFENQDKDIDLVSEKDVVSCPSNEATDVSTQS-ESNKGTQTKKIGRK human APMSNSPSKPSLPFQTSQSTGTEPFFKQKSLLLKQKQLNNSSVSSPQQNPWSNCKALPNS S.cerevisiae HADYHLALKLSEGLNGAEESSKNLSFGEKRLLFSRKRPNSQHTATPQKKQVTSSKNILSF . . . : :... : :. : :. .. . : :.: :. * : atRAD30 MNNSKEKNRGMPSIVDIFKNYNATPPSKQETQEDS---TVSSASKRAKLSSSSHN---SQ human LPTEYPGCVPVCEGVSKLEESSKATPAEMDLAHNSQSMHASSASKSVLEVTQKATPNPSL S.cerevisiae FTRKK------------------------------------------------------- : . atRAD30 VNQEVEESRETDWGYK-TDEIDQSVFDELPVEIQRELRSFLRTNKQFNTGKSK-GDGS-- human LAAEDQVPCEKCGSLVPVWDMPEHMDYHFALELQKSFLQPHSSNPQVVSAVSHQGKRNPK S.cerevisiae ------------------------------------------------------------ atRAD30 --------------TSSIAHYFPPLNR human SPLACTNKRPRPEGMQTLESFFKPLTH S.cerevisiae ---------------------------
Figure 3.19 Multiple protein alignment of REV3 homologues.
Conserved and conservatively replaced residues are indicated. The six
conserved sequence motifs thought to be characteristic of DNA polymerases
(Gibbs et al., 1998) are highlighted in the human protein in coloured italics, and
numbered above.
Accession Numbers of these entries are:
human Accession Number 3913527
mouse Accession Number 1304154
Arabidopsis Accession Number AC004393
S. cerevisiae Accession Number 118901
This alignment was generated using the CLUSTALW software package, in which
* denotes identical amino acids at that position, and : or . denotes conservative
replacement.
human MFSVRIVTADYYMASPLQGLDTCQSPLTQAPVKK ----1,180 intervening amino acids---- mouse ------------------------------------------------------------ Arabidopsis ------------------------------------------------------------ S.cerevisiae ------------------------------------------------------------ human VDDGKKKPRAKQKTNEKGTSRKHITLKDEKIKSQSGAEVKFVLKHQN-VSEFASSSGGSQ mouse ------------------------------------------------------------ Arabidopsis --------MADSQSGSNVFSLRIVSIDYYMASPIPGYNICYSSFQGSEVNEVPVIRIYGS S.cerevisiae ------------------------------------------------------------ Human LLFKQKDMPLMGSAVDHPLSASLPTGINAQQKLSG-CFSSFLESKKSVDLQTFPSS-RDD Mouse ------------------------------------------------------------ Arabidopsis TPAGQKTCLHIHRALPYLYIPCSEIPLEHHKGVDGSTLALSLELEKALKLKGNAASKRQH S.cerevisiae ------------------------------------------------------------ Human LHPSVVCNSIGPGVSKINVQRPHNQSAMFTLKESTLIQKNIFDLSNHLSQVAQNTQISSG Mouse ------------------------------------------------------------ Arabidopsis IHD---CEIVRAKKFYGYHSTEEAFVKIYLYPYSSYHPPDVARAASLLLAGAVLGKSLQP S.cerevisiae ------------------------------------------------------------ Human MSSKIEDNANNIQRNYLSSIGKLSEYRNSLESKLDQAYTPNFLHCKDSQQQIVCIAEQSK Mouse ------------------------------------------------------------ Arabidopsis YESHIPFILQFLVDYNLYGMGHVHISKMKFRSPVPHHFRPRRFDLDD------CPGQRID S.cerevisiae ------------------------------------------------------------ Human HSETCSPGNTASEESQMPNNCFVTSLRSPIKQIAWEQ-KQRGFILDMSNFKPERVKPRSL Mouse ------------------------------------------------------------ Arabidopsis EVAITKANSSAAASVSFP----VWSLSTIPGQWMWNLSEESDTPLSQSQHRHQHHYRRQS S.cerevisiae ------------------------------------------------------------
Human SEAISQTKALSQCKNRNVSTPSAFGEGQSGLAVLKELLQKRQQKAQNANTTQDPLSNKHQ Mouse ------------------------------------------------------------ Arabidopsis LCELEGDATSSDILNQQFKMYNSLSQAQSDTNMVQSLVAIWEEEYERTGVHDAPIP--PD S.cerevisiae ------------------------------------------------------------ Human PNKNISGSLEHNKANKRTRSVTSPRKPRTPRSTKQKEKIPKLLKVDSLNLQNSSQLDNSV Mouse ------------------------------------------------------------ Arabidopsis PGK------PSAADVLQTMSDYVGFGNMLKEMLNKVELSPPGMKPTAVSSAGPDMHAKPE S.cerevisiae ----------------------------------MSRESNDTIQSDTVRSSSKSDYFRIQ Human SDDSPIFFSDPGFESCYSLEDSLSPEHNYNFDINTIGQTGFCSFYSGSQFVPADQNLPQK Mouse ------------------------------------------------------------ Arabidopsis ITDLQALNHMVGTCSEFPASEQLSPLGEKSEEASMENDE--------YMKTPTDRDTPAQ S.cerevisiae LNNQDYYMSKP--TFLDPSHGESLPLNQFSQVPNIRVFG----------ALPTGHQVLCH Human FLSDAVQDLFPGQAIEKNEFLSHDNQKCDEDKHHTTDSASWIRSGTLSPEIFEKSTIDSN Mouse ------------------------------------------------------------ Arabidopsis IQDAEALGLFKWFASSQ---AAEDINSDDEILRETILSPLLPLASINKVLEMASTDYVSQ S.cerevisiae VHGILPYMFIKYDGQIT---DTSTLRHQRCAQVHKTLEVKIRASFKRKKDDKHDLAGDKL Human ENRRHNQWKNSFHPLTTRSNSIMDSFCVQQ---AEDCLSEKSRLNRSSVSKEVFLSLPQP Mouse ------------------------------------------------------------ Arabidopsis SQKECQDILDSQENLPDFGSSTKRALPSNP---DSQNLRTSS--DKQSLEIEVASDVPDS S.cerevisiae GNLNFVADVSVVKGIPFYGYHVGWNLFYKISLLNPSCLSRISELIRDGKIFGKKFEIYES Human NNSDWIQGHT-RKEMGQS--LDSANTSFTAILSSPDGELVDVACEDLELYVSRNNDMLTP Mouse ------------------------------------------------------------ Arabidopsis STSNGASENS-FRRYRKSDLHTSEVMEYKNRSFSKSNKPSNSVWGPLPFTLTKNLQKDFD S.cerevisiae HIPYLLQWTADFNLFGCSWINVDRCYFRSPVLNSILDIDKLTINDDLQLLLDR--FCDFK
Human TPDSSPRS-TSSPSQSKNGSFTPRTANILKPLMSPPSREEIMATLLDHDLSETIYQEPFC Mouse ------------------------------------------------------------ Arabidopsis STNASDKLGLTKISSYPMNEMTDNYIVPVKEHQADVCNTIDRNVLAGCSLRDLMRKKRLC S.cerevisiae CNVLSRRDFPRVGNGLIEIDILPQFIKNREKLQH---RDIHHDFLEKLGDISDIPVKPYV Human SNPSDVPEKPREIGGRLLMVETRLANDLAEFEGDFSLEGLRLWKTAFSAMTQNPRPGSPL Mouse ------------------------------------------------------------ Arabidopsis HGESPVSQHMK---SRKVRDSRHGEKNECTLRCEAKKQGPALSAEFSEFVCGDTPNLSPI S.cerevisiae SSARDMINELT---MQREELSLKEYKEPPETKRHVSGHQWQSSGEFEAFYKKAQHKTSTF Human RSGQGVVNKGSSNSPKMVEDKKIVIMPCKCAPSRQLVQVWLQAKEEYERSKKLPKTKPTG Mouse ------------------------------------------------------------ Arabidopsis DSGNCECNISTESSELHSVDR--------CSAKETASQNSDEVLRNLS-STTVPFGKDPQ S.cerevisiae DGQIPNFENFIDKNQKFSAIN---------TPYEALPQLWPRLPQIEINNNSMQDKKNDD Human VVKSAENFSSSVNPDDKPVVPPKMDVSPCILPTTAHTKEDVDNSQIALQAPTTGCSQTAS Mouse ------------------------------------------------------------ Arabidopsis TVESGTLVSSNIHVGIEIDSVQKSGREQESTANETDETGRLICLTLSKKPPSLDCLSAGL S.cerevisiae QVNASFTEYEICGVDNENEGVKGSNIKSRSYSWLPESIASPKDSTILLDHQTKYHNTINF Human -ESQMLPPVASASDPEKDEDDDDNYYISYSSPDSPVIPPWQQPISPDSKALNGDDRPSSP Mouse ------------------------------------------------------------ Arabidopsis -QDSAHSHEIHAREKQHDEYEGN----SN---DIPFFPLEDN--KEEKKHFFQGTSLGIP S.cerevisiae SMDCAMTQNMASKRKLRSSVSAN---------KTSLLSRKRK--KVMAAGLRYGKR--AF Human VEELPSLAFENFLKPIKDGIQKSPCSEPQEPLVISPINTRARTGKCESLCFHSTPIIQRK Mouse ------------------------------------------------------------ Arabidopsis LHHLNDGSNLYLLTPAFS---PPSVDSVLQWISNDKGDSNIDSEKQPLRDNHNDRGASFT S.cerevisiae VYGEPPFGYQDILNKLED--EGFPKIDYKDPFFSNPVDLENKPYAYAGKRFEISSTHVST
Human LLERLPEAPGLSPLSTEPKTQKLSNKKGSNTDTLRRVLLTQAKNQFAAVNTPQKETSQID Mouse ------------------------------------------------------------ Arabidopsis DLASASNVVSVSEHVEQHNNLFVNSESNAYTESEIDLKPKGTFLNLNLQASVSQELSQIS S.cerevisiae RIPVQFGGETVSVYNKPTFDMFS-SWKYALKPPTYDAVQKWYNKVPSMGNKKTESQISMH Human GPSLNNTYGFKVSIQNLQEAKALHEIQNLTLISVELHARTRRDLEPDPEFDPICALFYCI Mouse ------------------------------------------------------------ Arabidopsis GP-DGKSGPTPLSQMGFRDPASMGAGQQLTILSIEVHAESRGDLRPDPRFDSVNVIALVV S.cerevisiae TPHSKFLYKFASDVSGKQKRKKSSVHDSLTHLTLEIHANTRSDKIPDPAIDEVSMIIWCL Human SSDTPLPDTEKTELTGVIVIDKDKTVFSQDIRYQTPLLIRSGITGLEVTYAADEKALFHE Mouse -----------------------------------------------GTYAADEKALFQE Arabidopsis QNDD------------SFVAEVFVLLFSPDSIDQR---NVDGLSGCKLSVFLEERQLFRY S.cerevisiae EEET-------FPLDLDIAYEGIMIVHKASEDSTFPTKIQHCINEIPVMFYESEFEMFEA .* :*. IV Human IANIIKRYDPDILLGYEIQMHSWGYLLQRAAALS-IDLCRMISRVPDDKIENRFAAER-- Mouse ITNIIKRYDPDILLGYEIQMHSWGYLLQRAAALS-VDLCQMISRVPDDKIENRFAAER-- Arabidopsis FIETLCKWDPDVLLGWDIQGGSIGFLAERAAQLG-IRFLNNISRTPSPTTTNNSDNKRKL S.cerevisiae LTDLVLLLDPDILSGFEIHNFSWGYIIERCQKIHQFDIVRELARVKCQIKTKLS------ : : : ***:* *::*: * *:: :*. : . : . ::*. : Human ----------------------DEYGSYTMSEINIVGRITLNLWRIMRNEVALTNYTFEN Mouse ----------------------DDYGSDTMSEINIVGRITLNLWRIMRNEVGLTNYTFEN Arabidopsis GNNLLPDPLVANPAQVEEVVIEDEWGRTHASGVHVGGRIVLNAWRLIRGEVKLNMYTIEA S.cerevisiae ----------------------DTWGYAHSSGIMITGRHMINIWRALRSDVNLTQYTIES * :* * : : ** :* ** :*.:* *. **:*
Human VSFHVLHQRFPLFTFRVLS-DWFDNKTDLYRWKMVDHYVSRVRGNLQMLEQLDLIGKTSE Mouse VSFHVLHQRFPLFTFRVLS-DWFDNKTDLYRWKMVDHYVSRVRGNLQMLEQLDLIGKTSE Arabidopsis VSEAVLRQKVPSIPYKVLT-EWFSSGPAGARYRCIEYVIRRANLNLEIMSQLDMINRTSE S.cerevisiae AAFNILHKRLPHFSFESLTNMWNAKKSTTELKTVLNYWLSRAQINIQLLRKQDYIARNIE .: :*:::.* :.:. *: * . . ::: : *.. *:::: : * * :. * Human MARLFGIQFLHVLTRGSQYRVESMMLRIAKPMNYIPVTPSVQQRSQMRAPQCVPLIMEPE Mouse MARLFGIQFLHVLTRGSQYRVESMMLRIAKPMNYIPVTPSIQQRSQMRAPQCVPLIMEPE Arabidopsis LARVFGIDFFSVLSRGSQYRVESMLLRLAHTQNYLAISPGNQQVASQPAMECVPLVMEPE S.cerevisiae QARLIGIDFHSVYYRGSQFKVESFLIRICKSESFILLSPGKKDVRKQKALECVPLVMEPE **::**:* * ****::***:::*:.:. .:: ::*. :: . * :****:**** II Human SRFYSNSVLVLDFQSLYPSIVIAYNYCFSTCLGHVENLGKYDEFKFGCTSLRVPPDLLYQ Mouse SRFYSNSVLVLDFQSLYPSIVIAYNYCFSTCLGHVENLGKYDEFKFGCTSLRVPPDLLYQ Arabidopsis SAFYDDPVIVLDFQSLYPSMIIAYNLCFSTCLGKLAHL---KMNTLGVSSYSLDLDVLQD S.cerevisiae SAFYKSPLIVLDFQSLYPSIMIGYNYCYSTMIGRVREIN-LTENNLGVSKFSLPRNILAL * **...::**********::*.** *:** :*:: .: .:* :. : ::* VI Human VRHDITVSPNGVAFVKPSVRKGVLPRMLEEILKTRFMVKQSMKAYK-QDRALSRMLDARQ Mouse IRHDVTVSPNGVAFVKPSVRKGVLPRMLEEILKTRLMVKQSMKSYK-QDRALSRMLNARQ Arabidopsis LNQILQT-PNSVMYVPPEVRRGILPRLLEEILSTRIMVKKAMKKLTPSEAVLHRIFNARQ S.cerevisiae LKNDVTIAPNGVVYAKTSVRKSTLSKMLTDILDVRVMIKKTMNEIGDDNTTLKRLLNNKQ :.: : **.* :. ..**:. *.::* :**..*.*:*::*: .: .* *::: :* III I Human LGLKLIANVTFGYTSANFSGRMPCIEVGDSIVHKARETLERAIKLVNDTKKWGARVVYGD Mouse LGLKLIANVTFGYTAANFSGRMPCIEVGDSIVHKARETLERAIKLVNDTKKWGARVVYGD Arabidopsis LALKLIANVTYGYTAAGFSGRMPCAELADSIVQCGRSTLEKAISFVNANDNWNARVVYGD S.cerevisiae LALKLLANVTYGYTSASFSGRMPCSDLADSIVQTGRETLEKAIDIIEKDETWNAKVVYGD *.***:****:***:*.******* ::.****: .*.***:**.::: ..*.*:*****
Human TDSMFVLLKGATKEQSFKIGQEIAEAVTATNPKPVKLKFEKVYLPCVLQTKKRYVGYMYE Mouse TDSMFVLLKGATKEQSFKIGQEIAEAVTATNPRPVKLKFEKVYLPCVLQTKKRYVGYMYE Arabidopsis TDSMFVLLKGRTVKEAFVVGQEIASAITEMNPHPVTLKMEKVYHPCFLLTKKRYVGYSYE S.cerevisiae TDSLFVYLPGKTAIEAFSIGHAMAERVTQNNPKPIFLKFEKVYHPSILISKKRYVGFSYE ***:** * * * ::* :*: :*. :* **:*: **:**** *..* :******: ** V Human TLDQKDPVFDAKGIETVRRDSCPAVSKILERSLKLLFETRDISLIKQYVQRQCMKLLEGK Mouse TLDQKEPVFDAKGIETVRRDSCPAVSKILERSLKLLFETRDISLIKQYVHRQCMKLVEGK Arabidopsis SPNQREPIFDAKGIETVRRDTCEAVAKTMEQSLRLFFEQKNISKVKSYLYRQWKRILSGR S.cerevisiae SPSQTLPIFDAKGIETVRRDGIPAQQKIIEKCIRLLFQTKDLSKIKKYLQNEFFKIQIGK : .* *:************ * * :*:.::*:*: :::* :*.*: .: :: *: Human ASIQDFIFAKEYR-GSFSYKPGACVP-ALELTRKMLTYDRRSEPQVGERVPYVIIYGTPG Mouse ASIQDFIFAKEYR-GSFSYRPGACVP-ALELTRKMLAYDRRSEPRVGERVPYVIIYGTPG Arabidopsis VSLQDFIFAKEVRLGTYSTRDSSLLPPAAIVATKSMKADPRTEPRYAERVPYVVIHGEPG S.cerevisiae VSAQDFCFAKEVKLGAYKSEKTAPAG--AVVVKRRINEDHRAEPQYKERIPYLVVKGKQG .* *** **** : *::. . : :. : : * *:**: **:**::: * * Human VPLIQLVRRPVEVLQDPT-LRLNATYYITKQILPPLARIFSLIGIDVFSWYHELPRIHKA Mouse LPLIQLIRRPAEVLQDPT-LRLNATYYITKQILPPLARIFSLIGIDVFSWYQELPRIQKA Arabidopsis ARLVDMVVDPLVLLDVDTPYRLNDLYYINKQIIPALQRVFGLVGADLNQWFLEMPRLTRS S.cerevisiae QLLRERCVSPEEFLEGEN-LELDSEYYINKILIPPLDRLFNLIGINVGNWAQEIVKSKRA * : * .*: . .*: ***.* ::*.* *:*.*:* :: .* *: : :: Human TSSSR--SEPEGRKGTISQYFTTLHCPVCDDLTQHG----ICSKCRSQPQHVAVILNQEI Mouse TSSSR--SELEGRKGTISQYFTTLHCPVCDDLTQHG----ICSKCRSQPQHVAIILNQEI Arabidopsis SLGQRPLNSKNSHKTRIDYFYLSKHCILCGEVVQESA--QLCNRCLQNKSAAAATIVWKT S.cerevisiae STTTT-------KVENITRVGTSATCCNCGEELTKICSLQLCDDCLEKRSTTTLSFLIKK : : * : * *.: . :*. * .: . .: : :
Human RELERQQEQLVKICKNCTG---------CFDRHIPCVSLNCPVLFKLSRVNRELSKAP-- Mouse RELERKQEQLIKICRNCTG---------SFDRHIPCVSLNCPVLFKLSRVNRELSKAP-- Arabidopsis SKLEREMQHLATICRHCGGGDW------VVQSGVKCNSLACSVFYERRKVQKELRGLSSI S.cerevisiae LKRQKEYQTLKTVCRTCSYRYTSDAGIENDHIASKCNSYDCPVFYSRVKAERYLRDNQSV : ::: : * .:*: * . * * *.*::. :.:: * Human ------YLRQLLDQF Mouse ------YLRQLLDQF Arabidopsis ATESELYPKCMAEWF S.cerevisiae QREE--ALISLNDW- : :
Figure 4.1. Possible phylogenetic relationships between
RAD3/XPD homologues.
The plant protein is displayed as a completely separate entry, clearly
distinct from the fungal grouping, and the vertebrate grouping.
Accession numbers of these entries are:
human XPD Accession Number 11950
fish Xiphophorus XPD Accession Number 2134009
Arabidopsis atXPD Accession Number AF188623
S. pombe RAD15 Accession Number 1709995
S. cerevisiae RAD3 Accession Number 131812
This dendogram was generated by the CLUSTALW software package, using
default conditions.
Arabidopsis
S.pombe
S.cerevisiae
human
fish
Figure 4.a Growth curves for yeast rad3 mutants at 37oC.
This chart illustrates the differences in growth rates at 37oC of otherwise isogenic haploid rad3∆ strains carrying the rad3ts14
temperature-sensitive allele (‘rad3ts”), and with an expression vector carrying either wild-type RAD3 (“rad3ts/RAD3”) or atXPD
(“rad3ts/atXPD”).
Figure 4.b. Growth rates of rad3∆ recombinant strains
Strains were grown initially at 30oC (permissive temperature) to establish growth, then shifted to 37oC (restrictive
temperature) at Time = 0.
Figure 4.2. Possible phylogenetic relationships of RAD1/XPF protein
homologues.
This figure presents possible evolutionary relationships between
mammals, insects, fungi and plants.
Accession Numbers of these entries are:
mouse ERCC4 Accession Number 2896801
human XPF Accession Number 2842712
fly MEI9 Accession Number 3914026
N. crassa MUS38 Accession Number 3219304
S. cerevisiae RAD1 Accession Number 131810
atXPF-3 This study
This dendogram was generated by the CLUSTALW software package,
using default conditions.
fly
atXPF
N.crassa
S.cerevisiae
mouse
human
Figure 4.3 Multiple protein alignment of N, I and C regions of
RAD2/XPG homologues.
(a) Protein sequences corresponding to the conserved N region of
S. cerevisiae RAD2 protein.
(b) Protein sequences corresponding to the conserved I region of S.
cerevisiae RAD2 protein.
(c) Protein sequences corresponding to the conserved C region of S.
cerevisiae RAD2 protein.
Amino acids Asp-77 and Glu-791 (see section 4.5) are highlighted in bold
type.
Accession Numbers of these entries are:
mouse XPG Accession Number 549454
human XPG Accession Number 267420
atXPG this study
S. cerevisiae RAD2 Accession number 131811
This alignment was generated using the CLUSTALW software package, in
which * denotes identical amino acids at that position, and : or . denotes
conservative replacement.
(a) mouse MGVQGLWKLLECSGHRVSPEALEGKVLAVDISIWLNQALKGVRDSHGNVIENAHLLTLFH human MGVQGLWKLLECSGRQVSPEALEGKILAVDISIWLNQALKGVRDRHGNSIENPHLLTLFH atXPG MGVQCLWELLAPVGRRVSVETLANKRLAIDASIWMVQFIKAMRDEKGDMVQNAHLIGFFR S.cerevisiae MGVHSFWDIAGPTARPVRLESLEDKRMAVDASIWIYQFLKAVRDQEGNAVKNSHITGFFR ***: :*.: .: * *:* .* :*:* ***: * :*.:** .*: ::*.*: :*: mouse RLCKLLFFRIRPIFVFDGDAPLLKKQTLAKRRQRK human RLCKLLFFRIRPIFVFDGDAPLLKKQTLVKRRQRK atXPG RICKLLFLRTKPIFVFDGATPALKRRTVIARRRQR S.cerevisiae RICKLLYFGIRPVFVFDGGVPVLKRETIRQRKERR *:****:: :*:***** .* **:.*: *:.::
(b) mouse LKAQKQQQDRIAASVTGQMFLESQELLRLFGVPYIQAPMEAEAQCAVLDLSDQTSGTITD human LKAQKQQQERIAATVTGQMFLESQELLRLFGIPYIQAPMEAEAQCAILDLTDQTSGTITD atXPG -------------------------LLQIFGIPYIIAPMEAEAQCAFMEQSNLVDGIVTD S.cerevisiae ---QQMKDKRDSDEVTMDMIKEVQELLSRFGIPYITAPMEAEAQCAELLQLNLVDGIITD ** **:*** ********** : : ..* :** mouse DSDIWLFGARHVYKNFFNKNKFVEYYQYVDFYSQLGLDRNKLINLAYLLGSDYTEGIPTV human DSDIWLFGARHVYRNFFNKNKFVEYYQYVDFHNQLGLDRNKLINLAYLLGSDYTEGIPTV atXPG DSDVFLFGARSVYKNIFDDRKYVETYFMKDIEKELGLSRDKIIRMAMLLGSDYTEGIRGI S.cerevisiae DSDVFLFGGTKIYKNMFHEKNYVEFYDAESILKLLGLDRKNMIELAQLLGSDYTNGLKGM ***::***. :*:*:*...::** * .: . ***.*.::*.:* *******:*: : mouse GCVTAMEILNEFPGRGLDPLLKF human GCVTAMEILNEFPGHGLEPLLKF atXPG GIVNAIEVVTAFPE--EDGL--- S.cerevisiae GPVSSIEVIAEFGN--LKNF--- * *.::*:: * . :
(c) atXPG -------------GRSKKTK- S.cerevisiae KKLNTSKRISTATGKLKKRKM mouse AKLIKSHRLSRAVTCMLRKER human AKRIKSQRLNRAVTCMLRKEK : :
Table 1.1 Homologous repair and/or transcription proteins.
S. cerevisiae S. pombe Human Suggested Function RAD1
RAD2
RAD3
RAD4
RAD10
RAD14
RAD23
RAD25/SSL2
RAD26
RAD28
RPA
TFB1
TFB2
TFB4
SSL1
POL δ
POL ε
PCNA
CDC9
RAD16
RAD13
RAD15
SWI10
ERCC3Sp
XPF/ERCC4
XPG/ERCC5
XPD/ERCC2
XPC
ERCC1
XPA
HHR23A/B
XPB/ERCC3
CSB
CSA
P62
P52
P34
P44
DNA Ligase I
With RAD10 as a 5’ endonuclease
3’ endonuclease
5’→3’ Helicase
Lesion binding
With RAD1 as a 5’ endonuclease
Early steps of NER
Lesion binding
3’→5’ Helicase
Inhibition of transcription
Early steps of NER
TFIIH subunit
TFIIH subunit
TFIIH subunit
Stimulates XPD in TFIIH
Replicative/repair polymerase
Replicative/repair polymerase
Processivity factor for polymerases
DNA ligation
Table 1.2. Potential DNA repair defective mutants of Arabidopsis thaliana.
Radiation sensitivities
Ultraviolet (UV)b
Mutant designationa
Progenitor line
Darkd Lighte γ γ γ γ radiationc
Ref.
rad1, rad2,rad3 Landsberg errecta UVC: sl, – s, ++ 1
rad4, rad5 Colombia UVC: sl, – s, ++ 1
uvh1-1 Colombia C10 UVB: UVC:
sl, sl,
++; ++;
r, r,
++ ++
UVB: UVC:
sl, sl,
+; +;
r, r,
++ ++
s, ++ 2
uvh3 Colombia UVB: UVC:
sl, sl,
++; ++;
r, r,
++ ++
UVB: UVC:
sl, sl,
++ ++
s, + 3
uvh4 Colombia UVB: UVC:
sl, sl,
+; +;
r, r,
– –
UVB: UVC:
sl, sl,
+ +
s, – 3
uvh5 Colombia UVB: UVC:
sl, sl,
+; +;
r, r,
– –
UVB: UVC:
sl, sl,
+ +
s, + 3
uvh6 Colombia UVB: UVC:
sl, sl,
++; ++;
r, r,
+ +
UVB: UVC:
sl, sl,
+ +
s, – 3
uvr1 L. errecta tt5 UVB r, ++ s, + 4
uvr2f uvr1 UVB r, ++ UVB: ++ (r)
r, ++ 4
uvr2–1 L. errecta UVB sl, +; r, + UVB: sl, +; r, + 4
uvr3f uvr1 UVB r, ++ UVB: ++ (r)
r, ++ 4
uvr5 L. errecta UVB r, ++ s, +; r, – 4
uvr6 L. errecta UVB r, – s, –; r, – 4
uvr7 L. errecta UVB r, ++ s, +; r, – 4
Table 1.2 (continued)
Radiation sensitivities
Ultraviolet (UV)b
Mutant designationa
Progenitor line
Darkd Lighte γ γ γ γ radiationc
Ref.
uvs1, uvs2, uvs6
Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ –;
r,
++
s, + 5
uvs3 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ –;
r,
+
s, ++ 5
uvs4 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
– ++;
r,
++
s, + 5
uvs5 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
++ +;
r,
++
s, + 5
uvs7 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ +;
r,
++
s, – 5
uvs8 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
– +;
r,
+
s, – 5
uvs9 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ –;
r,
++
s, – 5
uvs10 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ ++;
r,
+
s, – 5
uvs11 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
++ –;
r,
++
s, – 5
uvs12, uvs19, uvs21, uvs46
Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ –;
r,
–
s, + 5
uvs13 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
+ ++;
r,
–
s, + 5
uvs14 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
++ –;
r,
++
s, + 5
Table 1.2 (continued)
Radiation sensitivities
Ultraviolet (UV)b
Mutant designationa
Progenitor line
Darkd Lighte γ γ γ γ radiationc
Ref.
uvs15 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
+ +;
r,
–
s, + 5
uvs16 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
+ +;
r,
–
s, – 5
uvs17 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
++ –;
r,
–
s, + 5
uvs18, uvs32, uvs48
Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ +;
r,
–
s, + 5
uvs20, uvs24 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
+ –;
r,
–
s, – 5
uvs22 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
++ +;
r,
–
s, – 5
uvs23 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
– –;
r,
–
s, – 5
uvs25 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
– +;
r,
–
s, + 5
uvs26, uvs42 Wassilewskija UVC: r, – UVB: UVC:
sl, sl,
+ –;
r,
–
s, – 5
uvs27, uvs28 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
++ +;
r,
–
s, – 5
uvs30, uvs43, uvs44
Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ +;
r,
–
s, – 5
uvs31 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
– +;
r,
–
s, + 5
Table 1.2 (continued)
Radiation sensitivities
Ultraviolet (UV)b
Mutant designationa
Progenitor line
Darkd Lighte γ γ γ γ radiationc
Ref.
uvs33 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
– +;
r,
–
s, ++ 5
uvs34, uvs41 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
+ –;
r,
–
s, + 5
uvs35 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
– –;
r,
–
s, + 5
uvs36, uvs37, uvs38, uvs47, uvs49, uvs55
Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ +;
r,
–
s, + 5
uvs39 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ ++;
r,
–
s, – 5
uvs40 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ –;
r,
–
s, – 5
uvs45 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ +;
r,
–
s, ++ 5
uvs50 Wassilewskija UVC: r, ++ UVB: UVC:
sl, sl,
– –;
r,
–
s, ++ 5
uvs51 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
– –;
r,
–
s, – 5
uvs52 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
++ ++;
r,
–
s, + 5
uvs53 Wassilewskija UVC: r, + UVB: UVC:
sl, sl,
+ –;
r,
–
s, ++ 5
a The allelic relationships between these mutants have not all been
established. b Sensitivities are expressed in the form ‘Radiation source: plant material affected
(where s = seeds, sl = seedlings & r = roots), sensitivity’ (++ denotes >50% more
sensitive than wild-type, + denotes ~50% more sensitive than wild-type, – denotes
wild-type levels of resistance; wild-type in each case refers to the original
progenitor strain). c Symbols used are the same as for UV sensitivity. d Irradiation and incubation conditions that prevent photoreactivation. e Irradiation and incubation conditions that allow photoreactivation. f Sensitivity to UV in ‘dark’ conditions is because strains are derived from uvr1
and so are double mutants. Sensitivities are in comparison to L. errecta tt5.
References are: 1, (Davies et al., 1994); 2, (Harlow et al., 1994); 3, (Jenkins et al.,
1995); 4, (Jiang et al., 1997); 5, (Vonarx, 1998).
Table 1.3 Characterised plant DNA repair genes or cDNAs.
Gene/c-DNA Plant Accessiona Potential Function Referenced
aMAG Atb 429157 BER 1
DRT100 At 544134 Recombination 2
DRT101 At 1169198 Excision Repair 3
DRT102 At 1169199 Excision Repair 3
DRT111 At 166694 Recombination 4
DRT112 At 166696 Recombination 4
Ligase At 1359495 Ligation 5
PHH1 At 1468977 Photoreactivation 6
PHR2 At 1617219 Photoreactivation 7
UVR3 At BAA24449 Photoreactivation 8
ERCC1 Lily AJ002990 NER/Recombination 9
atXPB At U29168 NER/Transcription 10
atRAD1 At AF089003 NER/Recombination 11
UVH1 At AF160500 NER/Recombination 12
RAD23 At AAF32461 NER/Transcription 13
RAD23 Osc 1488297 NER/Transcription 14
RAD23-I Carrot T14336 NER/Transcription 15
RAD23-II Carrot TAA72742 NER/Transcription 15
RAD51 At 1706949 Ds break repair 16
RECA At 2500098 Recombination 17
UBC1 At 1076424 Ubiquitination 18
UBC2 At 2689243 Ubiquitination 18
UBC3 At 431262 Ubiquitination 18
atMLH1 At CAA10163 MMR 19
atMSH2 At 024617 MMR 20
atMSH3 At 065607 MMR 21
atMSH6 At O04716 MMR 22
a Database Accession number. b Arabidopsis thaliana. c Oryza sativa (rice). d References are: 1, (Santerre and Britt, 1994); 2, (Pang et al., 1992); 3, (Pang et al.,
1993a); 4, (Pang et al., 1993b); 5, (Taylor et al., 1996); 6, (Hoffman et al., 1996); 7, (Taylor et al., 1996); 8, (Nakajima et al., 1998); 9, (Xu et al., 1998); 10, (Ribiero et al., 1998); 11, (Gallego et al., 2000); 12, (Liu et al., 1999); 13, (Lin et al., 2000); 14, (Schultz and Quatrano, 1997); 15, (Sturm and Lienhard, 1998); 16, (Urban et al., 1996); 17, (Cerutti et al., 1992); 18, (Sullivan et al., 1994); 19, (Jean et al., 1999); 20, (Culligan and Hays, 1997); 21, (Bevan et al., 1998); 22, (Till et al., 1997).
Table 2.1. PCR and sequencing primers used in this investigation
Primer Name Amino Acid Sequencea Primer Nucleotide Sequenceb
RAD3-F1 EMPSGTG GARATGCCNWSNGGNACNGG
RAD3-R1 DEAHNID RTCDATRTTRTGNGCYTCRTC
AtmRNA-RAD3-F1 FLRKMAQP GTTGAGCCATTTTCCGAAG
AtmRNA-RAD3-F2 DMAY#c TTTACTAATAAGCCATGTCT
AtmRNA-RAD3-R1 MIFKIE AAGATGATCTTTAAAATCGAA
AtmRNA-RAD3-SF1 KLVYCTR GCTTGTTTACTGTACTCG
AtmRNA-RAD3-SF2 LLPPGV GTTGTTGCCTCCTGGTG
AtmRNA-RAD3-SF3 RRVTL GAGGAGGGTTACACTTG
AtmRNA-RAD3-SF4 SFVSSL GCTTTGTATCGTCACTG
AtmRNA-RAD3-SF5 LYPRLL CTTTACCCCCGTCTTCTT
AtmRNA-RAD3-SF6 TWNETGI ATGGAATGAAACTGGAAT
AtmRNA-RAD3-SF7 ILRARLE TATTACGAGCAAGATTGG
AtmRNA-RAD3-SR1 KVAEGI GATACCTTCAGCAACTTTC
AtmRNA-RAD3-SR3 CYDRLQ ACTGGAGCCGGTCATAAC
ScRAD3-F2 TMKFYID ACAATGAAGTTTTATATAGATG
ScRAD3-R1 DEDIEMQ CTGCATTTCTATATCTTCATC
RAD3-Cap LVYCTRTV GCTTGTTTACTGTACTCGTACTGT
RAD3-5’Block TSYRLSRPDSPIK TCACCAGCTATCGACTCTCTCGTCCTGATTCTCCGATCAA
Table 2.1 continued
Primer Name Amino Acid Sequencea Primer Nucleotide Sequenceb
RAD3-3’Block HEMEKTLGELKLL CCACGAGATGGAGAAAACGTTAGGTGAGCTTAAGCTACT
AtmRNA-RAD1-F1 IQDLFQS GCTCTGGAAAAGATCTTGAA
AtmRNA-RAD1-R1 MALKYH ATGGCGCTGAAATATCACC
AtmRNA-RAD1-F7 YPTLL# TCATAGCAAAGTCGGATAC
AtmRNA-RAD1-SR10 LPNVLH TCTACCAAATGTTCTTCACC
AtmRNA-RAD1-F2 STEFPAS GAAGCTGGAAACTCTGTAG
AtmRNA-RAD1-SF3 TIQNSEG CCTTCCGAGTTCTGTATTG
AtmRNA-RAD1-SF5 EEAPKW CATTTTGGTGCCTCTTCC
gRAD1-F1 LEREP GGTTCTCGCTCCAATTCCTGA
AtmRNA-RAD1-SF7 LHSPS GCGACGGAGAGTGAAGG
Gal1 Fwd Vector Primer TTATATGGAGATATGAAATTGCAG
V5 C-term Rev Vector Primer ACCGAGGAGAGGGTTAGGGAT
AtmRNA-RAD1-SR2 TQRIP GACTCAACGGATTCCAG
AtmRNA-RAD1-SR3 DDVDLT TGATGTTGATGATTTGACTG
AtmRNA-RAD1-SR4 TNVATG CAAATGTGGCTACTGGCG
AtmRNA-RAD1-SR6 MASGNN ATGGCTTCTGGAAATAAC
AtmRNA-RAD1-SR7 IIVYHP TCATTGTCTACCATCCT
AtmRNA-RAD30-F1 MPV CTGAAATAGAAATGCCGG
AtmRNA-RAD30-R1 PPLNR# ATCTATCTATTCAATGGTTGG
AtmRNA-RAD30-R3 TVSSASK TTACTGGCTGATGACACTG
Table 2.1 continued
Primer Name Amino Acid Sequencea Primer Nucleotide Sequenceb
AtmRNA-RAD2-F1 MGVQG AAAATGGGAGTACAAGGTC
AtmRNA-RAD2-F2 EAEAQC GAAGCAGAGGCACAGTG
AtmRNA-RAD2-F3 ADASIW GCAGATGCGAGTATATGG
AtmRNA-RAD2-R1 SKKTK# TTATTTCGTTTTCTTGGACC
AtmRNA-RAD2-R2 EAEAQC CACTGTGCCTCTGCTTC
pNF1000-SF1 LEPFSER TTAGAACCTTTTAGTAAACG
pNF1000-SR1 LKLGRE CTCACGTCCTAATTTCAAC
AP1 Not applicable AGTCATGCCCCGCGCCCATTTTTTTTTTTTTTTTT
Ent100-6 Not applicable ATAGTCATGCCCCGCGCCCA
AtmRNA-REV3-SF1 GVHVGG GTGTTCATGTTGGAGGTAG
AtmRNA-REV3-SF2 LRLAHT CTGAGATTAGCACATACAC
AtmRNA-REV3-SF3 LLEEI GCTGCTAGAGGAGATTC
AtmRNA-REV3-SF4 VVGQEI GTAGTCGGACAAGAGATT
AtmRNA-REV3-SF5 LPPAAI TTCCTCCAGCAGCTATTG
AtmRNA-REV3-SF6 VQESAQL TTCAAGAATCTGCTCAACTA
AtmRNA-REV3-F4 5’ untranslated CGCCAAATCATAATTCGTG
AtmRNA-REV3-F5 LEMASTD TTGAAAGGCTAGTACAGAT
AtmRNA-REv3-F7 EERQLF AAGAGAGGCAACTATTCAG
AtmRNA-REV3-R4 EWF# CGTCAAGGTTAGAACCACTCAG
AtmRNA-REV3-R5 PDFGSST CGAACTCCCAAAGTCTG
Table 2.1 continued
Primer Name Amino Acid Sequencea Primer Nucleotide Sequenceb
AtmRNA-REV3-R6 NPAQVE TTCTACTTGAGCAGGATTA
AtmRNA-REV3-R7 VPLVMEP GGTTCCATCACAAGAGGTA
AtmRNA-REV3-R8 Unknown GAAAGAAGAGAAGGTGACTAC
AtmRNA-REV3-F8 LSSTTVP TTGTCATCTACCACTGTTCC
a Amino acid sequence is the translation of the coding strand within the primer target sequence region. Amino acids are
represented by the IUPAC single letter code. b Nucleotides are represented by the standard IUPAC code (N=A+G+C+T; R=A+G; Y=C+T; S=C+G; D=A+T+G; W=A+T).
All primer sequences are written 5’→3’. c STOP codon.
Table 3.1 AtXPD exon/intron sizes
Exon No. Size (bp) Intron No. Size (bp)
1 537 1 126
2 281 2 392
3 83 3 98
4 404 4 132
5 194 5 87
6 181 6 152
7 126 7 196
8 89 8 105
9 78 9 93
10 78 10 287
11 109 11 97
12 120
Totals (bp) 2,280 1,765
Means (bp) 190 161
SD 147 97
Table 3.2. atXPF-3 exon/intron sizes.
Exon No. Size (bp) Intron No. Size (bp)
1 866 1 78
2 224 2 96
3 86 3 170
4 149 4 113
5 738 5 261
6 111 6 84
7 262 7 97
8 224 8 88
9 208
Totals (bp) 2867 987
Means (bp) 319 123
SD 282 63
Table 3.3 Variable intron/exon boundaries and their utilization in atXPF clones.
atXPF Clonea
No. Location Intronic sequence Exonic sequence Intronic sequence 1 2 3
I Intron 5/exon 6 AAUUAG GAUGGG + - +
II Intron 5/exon 6 UUUCAG UUUCUU - + -
III Exon 7/intron 7 CAUCAG GUCGUU + - -
IV Exon 7/intron 7 UUUCAG GUACCC - + +
V Consensusb U63nC6A100G100b G55U43
b
VI Consensus C36A62G78b G100U99A68A57
b
All sequences are written 5’ to 3’.
a Utilization of that junction in creation of that mRNA is indicated by a + sign. A – sign denotes that junction is not processed in that clone
b Consensus A. thaliana mRNA boundary sequences (Brown et al., 1996). Subscripts indicate percentage frequency.
Table 3.4 Resolution of disputed bases in RAD1.
Base Number a YSCRAD1 YSCRAD1A This Study Amino Acid Alteration
2648 A G G Y/C
2658 A G G Y/C
2688 A G G No change
2801 - A A Change of frame
2841/2 T - - Change of frame
3046 A G G R/G
a Base number refers to the position of the base in the genomic sequence.
Table 3.5 Codon usage in atXPD.
AAa Codon Freq
in this
CDS
% freq
in this
codon
% freq
in At
AA Codon Freq
in this
CDS
% freq
in this
CDS
% freq
in At
AA Codon Freq
in this
CDS
% freq
in this
CDS
% freq
in At
AA Codon Freq
in this
CDS
%freq
in this
CDS
% freq
in At
F TTT 18 58 52 S TCT 11 26 28 Y TAT 14 52 53 C TGT 8 50 60
F TTC 13 42 48 S TCC 4 9 12 Y TAC 13 48 47 C TGC 8 50 40
L TTA 10 12 14 S TCA 8 18 20 STOP TAA 0 0 38 STOP TGA 0 0 42
L TTG 18 21 22 S TCG 2 5 10 STOP TAG 1 100 21 W TGG 5 100 100
L CTT 21 25 26 P CCT 18 65 38 H CAT 13 76 62 R CGT 9 16 17
L CTC 10 12 17 P CCC 2 7 11 H CAC 4 24 38 R CGC 3 6 7
L CTA 12 14 11 P CCA 4 14 34 Q CAA 13 52 56 R CGA 11 20 11
L CTG 13 16 10 P CCG 4 14 17 Q CAG 12 48 44 R CGG 10 18 9
I ATT 19 49 41 T ACT 16 39 34 N AAT 19 79 52 S AGT 4 9 16
I ATC 12 31 35 T ACC 6 15 20 N AAC 5 21 48 S AGC 14 33 13
I ATA 8 20 24 T ACA 17 41 31 K AAA 14 37 49 R AGA 9 16 35
S/M ATG 21 100 100 T ACG 2 5 15 K AAG 24 63 51 R AGG 13 24 21
V GTT 29 51 40 A GCT 24 45 43 D GAT 34 72 68 G GGT 13 32 34
V GTC 5 9 19 A GCC 9 17 16 D GAC 13 28 32 G GGC 4 10 14
V GTA 13 23 15 A GCA 13 25 27 E GAA 16 38 52 G GGA 16 39 37
V GTG 10 17 26 A GCG 7 13 14 E GAG 26 62 48 G GGG 8 19 15
a Amino acids are represented by the IUPAC single-letter code. atXPD codon usage data was generated at the “countcodon” website
(http://www.kazusa.or.jp/codon/cgi).
Table 3.6 Codon usage in atXPF.
AAa Codon Freq
in this
CDS
% freq
in this
CDS
% freq
in At
AA Codon Freq
in this
CDS
% freq
in this
CDS
% freq
in At
AA Codon Freq
in this
CDS
% freq
in this
CDS
% freq
in At
AA Codon Freq
in this
CDS
% freq
in this
CDS
% freq
in At
F TTT 22 63 52 S TCT 26 25 28 Y TAT 15 63 53 C TGT 3 33 60
F TTC 13 37 48 S TCC 20 19 12 Y TAC 9 37 47 C TGC 6 66 40
L TTA 16 16 14 S TCA 15 14 20 STOP TAA 0 0 38 STOP TGA 1 100 42
L TTG 16 16 22 S TCG 13 13 10 STOP TAG 0 0 21 W TGG 6 100 100
L CTT 21 20 26 P CCT 17 38 38 H CAT 11 69 62 R CGT 8 14 17
L CTC 23 22 17 P CCC 5 11 11 H CAC 5 31 38 R CGC 5 9 7
L CTA 16 16 11 P CCA 14 31 34 Q CAA 17 49 56 R CGA 7 13 11
L CTG 10 10 10 P CCG 9 20 17 Q CAG 18 51 44 R CGG 7 13 9
I ATT 20 32 41 T ACT 17 35 34 N AAT 21 62 52 S AGT 17 17 16
I ATC 24 39 35 T ACC 8 16 20 N AAC 13 38 48 S AGC 12 12 13
I ATA 18 29 24 T ACA 18 37 31 K AAA 37 51 49 R AGA 17 30 35
S/M ATG 23 100 100 T ACG 6 12 15 K AAG 35 49 51 R AGG 12 21 21
V GTT 34 53 40 A GCT 26 46 43 D GAT 34 74 68 G GGT 16 40 34
V GTC 8 13 19 A GCC 9 16 16 D GAC 12 26 32 G GGC 8 20 14
V GTA 8 13 15 A GCA 16 28 27 E GAA 43 55 52 G GGA 11 28 37
V GTG 14 21 26 A GCG 6 10 14 E GAG 35 45 48 G GGG 5 12 15
a Amino acids are represented by the IUPAC single-letter code. atXPF codon usage data was generated at the “countcodon” website
(http://www.kazusa.or.jp/codon/cgi).
Table 4.1 Sequenced or putative plant Nucleotide Excision Repair genes
Gene or
c-DNA
Plant Species Database
Accessiona
Potential Function Reference
ERCC1 L. longiflorum AJ002990 NER/Recomb repair 1
atERCC1 Atb Unpublished NER/Recomb repair This study
atXPB At U29168 NER/Transcription 2
atXPD At AF188623 NER/Transcription This study
atXPF At AF191494 NER/Recombination This study
atRAD1 At AF089003 NER/Recombination 3
UVH1 At AF160500 NER/Recombination 4
atXPG At Unpublished NER/Transcription This study
RAD23 At AAF32461 NER/TCR 5
RAD23 Osc 1488297 NER/TCR 6
RAD23-I Carrot T14336 NER/TCR 7
RAD23-II Carrot TAA72742 NER/TCR 7
a Entrez (protein) Accession Number. b Arabidopsis thaliana. c Orysa sativa References are: 1, Xu et al., 1998; 2, Ribiero et al., 1998; 3, Gallego et al., 2000; 4, Liu et al., 2000; 5, Lin et al., 2000; 6, Schultz and Quatrano, 1997; 7, Sturm and Lienhard, 1998.
