CHAPTER 1 Mechanisms of Mutation - Elsevier · CHAPTER 1 Mechanisms of Mutation 5 mutagenic characteristics of such compounds. A major development was the discovery that metabolic

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CHAPTER 1

Mechanisms of Mutation

BERNARD S. STRAUSS

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA

INTRODUCTION

Mutation is a sudden, inheritable change in the genome [1] . The change needs to be sudden, that is, the change must be present in one cellular generation but not in the preceding generation. Mutations were originally defi ned as necessa-rily involving the gametes, but somatic mutations are now recognized as an important process. The phenomenon of recombination will often couple two sequences to give a molecule with such novel properties that it appears to have been a mutation, but geneticists have not usually classi-fi ed such events as mutations. As will be seen in the dis-cussion of chromosome aberrations (below) this separation between recombination and mutation is not clear-cut. The change also needs to be transmitted. An alteration in the DNA structure that prevents its replication is an inactivation, but not a mutation. On the other hand there are reversible changes possible in the structure, but not the coding prop-erties of the nucleotides, for example, cytosine or adenine methylation. These changes can be inherited, but since the pairing properties of the nucleotide are not altered they are considered as epigenetic change, notwithstanding the large effect such modifi cations can have on function. Mutations may involve single nucleotides, in which case we speak of point mutations. A few or many nucleotides may be added or deleted. Whole genes or groups of genes may be deleted, duplicated or moved to other chromosomes, or whole chro-mosomes may be added or lost as a result of errors in cell division. All such changes may have drastic effects on the life of the organism. On the other hand, many changes in the DNA have no discernible effect on function. As long as the interest of geneticists concentrated on Mendelian traits (or phenotypes), attributable to the action of a single gene, it was relatively simple to distinguish functionally signifi cant mutations. The discovery that humans may dif-fer at approximately one nucleotide in every thousand or, given a genome size of about three billion, at three million possible sites, the single nucleotide polymorphisms or SNPs

complicate the attempt to determine which changes are sig-nifi cant [2] . An additional complexity comes from the dis-coveries that differences in sequence statistically associated with different diseases are often found to be located in gene deserts, regions in which there are no known genes [3] .

Although only about 25,000 protein coding genes have been recognized, these account for less than 5% of the total DNA. However, it appears that almost half (50%) of the total DNA is transcribed. At least some of these tran-scripts have a function. Up to one thousand are processed into small (20 – 23 nucleotide) sequences called microRNA (miRNA) with key roles in regulating a variety of cell func-tions. The key players in this regulation are nucleotides 2 to 8 of the miRNA, the so-called “ seed region ” [4, 5] .

Insofar as the sequences coding these miRNAs have a physiological effect on the organism, they need to be consid-ered as genes in the same sense as protein coding genes. The DNA giving rise to these miRNAs is subject to mutation by the same mechanisms for base change discussed in the chap-ter. In addition, “ RNA editing ” changes the pairing specifi -city of sequences by conversion of particular adenosines (A) to inosine (I) by deamination. It is conceivable that alter-ations in the editing system could result in major changes in the function of particular miRNAs. Recently two point mutations in the seed region of one of the miRNA genes, miR-96, have been shown to segregate in a Mendelian way and to be responsible for a form of deafness [6] . It is likely that single base changes in other miRNA genes will be iden-tifi ed with other cases of inherited disease.

The total amount of DNA involved in coding for micro-RNAs is small, even though the initial transcripts before processing are several kilobases long. Even 1000 several kilobase genes would only constitute about 0.1% of a total genome of 3.3 � 10 9 bases. However, we may yet fi nd out that the 45% or so of transcribed DNA not accounted for, but mutable as a result of the processes described above, has some function. We are still at the beginning of our understanding of the structure of the human genome.

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Genetic Diagnosis of Endocrine Disorders4

POINT MUTATIONS

We know more about the mechanisms by which point muta-tions occur and of their effects than any of the other changes ( Table 1.1 ). The original association of a single point muta-tion with a particular disease was the discovery that sickle cell anemia was, in Pauling’s words, a “ molecular disease ” since all of its signs can be traced to a substitution of a glutamic acid by a valine in the hemoglobin molecule. This change was later shown to be due to a change from GAG (glutamic acid) to GTG (valine) [7] . The association of genetic change with a particular disease dates back at least a century to Garrod’s discovery of the Mendelian inherit-ance of alkaptonuria, but Garrod had no idea of the nature of the genetic change. The second reason is methodological. Before the age of PCR and of DNA sequencing, mutations were most easily studied in the bacteria and the bacterial viruses. Ernst Freese and Seymour Benzer fi rst systema-tized the possible single base changes. These investigators and their co-workers coined the terms “ transition ” to denote the change from one purine to another or of one pyrimidine to another. The four possible transitions are cytosine (C) to thymine (T) and its reverse, and adenine (A) to guanine (G) and its reverse. Freese and Benzer defi ned “ transversions ” as changes from a purine to a pyrimidine or the reverse. So a change from an A or a G to a C or a T, and the reverse C or T to A or G were defi ned as transversions. Shortly afterwards, Brenner pointed out that some of the putative transversions were actually additions or deletions of one or two nucleotides [8] . These were later called frameshifts, because of the discovery that the genetic code was formed of triplets, groups of three nucleotides read together to specify a particular amino acid. The addition (or deletion) of any number of nucleotides not divisible by three would result in a change in the reading “ frame ” of three nucle-otides at a time, thereby changing the amino acid composi-tion of all amino acids downstream of the coding change. The details of the genetic code, as elucidated in the 1960s, also indicated that such frameshifts might not only result in major changes in the amino acid composition of a protein but might also produce unexpected termination codons as a result of the shift. Point mutations that resulted in protein terminations were at fi rst termed “ nonsense ” mutations as opposed to missense mutations that resulted in the substi-tution of one amino acid for another. The nonsense muta-tions did not make “ sense ” , i.e. did not specify any amino acid. There are three such codons, now called termination (ter) codons: UAA, UAG, and UGA. Since messenger RNA is the molecule that is actually read by the protein synthesizing machinery, the code is an RNA code with U(racil) substituted for T(hymine). One of the stop codons, UGA, is read as tryptophan in mitochondria and the mito-chondrial code includes a few other variations: AGG and AGA are mitochondrial stop codons instead of coding for arginine, and AUA codes for methionine instead of

isoleucine. There are 64 possible codons but only 20 (natu-ral) amino acids and the code is degenerate, or redundant, in that several codons can specify the same amino acid. Point mutations within genes that do not change the mean-ing (amino acid coded) of the codon are termed synony-mous or “ silent ” as opposed to non-synonymous changes. Although there was some initial confusion about the neces-sity of punctuation between the triplet codons, it was real-ized that if reading of the code began at a fi xed site, and if the reading “ frame ” was designed to read three nucleotides at a time, the correct sequence of amino acids would auto-matically be produced.

This terminology was developed before it was realized that there were large amounts of non-coding DNA. Even though it was (is) used, “ frameshift ” has no meaning for mutations within such non-coding regions. A more recent term for small in sertions or del etions, regardless of their physiological effect is “ indel ” . Notwithstanding, or because of the universal nature of the code, there still remain some mysteries. The codon AUG specifi es methionine and it also signals “ start ” , but not always. UGA specifi es stop but some UGA codons specify the 21st amino acid, selenocysteine. The recoding of UGA is determined by the surrounding sequence, a characteristic stem loop located in the 3 � -end of mammalian mRNAs.

MUTAGENIC AGENTS

It was fi rst assumed that the fi delity of normal replica-tion stemmed from the stability of the A:T and G:C base pairs resulting from hydrogen bonding. The success of the early workers on the molecular nature of mutations came from their ability to account for the specifi city of a vari-ety of mutagenic base analogs and other mutagenic agents. They were able to draw acceptable alternative base pair-ings resulting from the incorporation of these compounds into DNA, or by their distortion of the replication machin-ery. For a number of years research on mutational mecha-nisms consisted largely of formulations of how changes in DNA structure resulting from treatment with, or incorpora-tion of, mutagenic chemicals could change the base pairing properties of the replicating DNA so that mistakes were made. The supposition by Bruce Ames that “ Carcinogens are Mutagens ” [9] (which did not, it turned out, mean that all carcinogens are mutagens) prompted chemists to study the changes in DNA structure produced by reaction with chemical and physical mutagens. Alkylating agents such as methyl nitrosourea and the chemotherapeutically active mustard gas derivatives were shown to react with individ-ual nucleotides to produce multiple changes. Production of O 6 methyl guanine by agents such as methyl nitroso-urea or methyl nitronitrosoguanidine was shown to promote mistaken base pairing, making understandable the highly


CHAPTER 1 ● Mechanisms of Mutation 5

mutagenic characteristics of such compounds. A major development was the discovery that metabolic systems in the host activated ingested compounds making it possi-ble for them to react with DNA. Carcinogenic polycyclic

hydrocarbons and afl atoxins are converted to epoxide derivatives with the participation of the cytochrome p450 system. These epoxides react directly with DNA produc-ing mutagenic adducts. The Ames assay, a bacterial test for

TABLE 1.1 Defi nitions

Base excision repair (BER) A repair mechanism in which single nucleotide bases are removed and replaced by a patch of one or at most a few nucleotides.

Copy number variation (CNV) Altered number of copies of a gene present in the genome. Deletion A loss of genetic material from a chromosome, often used to refer to a loss extensive enough to

be cytologically visible. DNA glycosylase An enzyme which recognizes and removes specifi c bases from DNA without breaking the

backbone chain. Frameshift mutation The addition or deletion of a number of nucleotides not divisible by 3, properly speaking in a

coding region of a gene. Largely replaced by the term “ indel ” . Hoogsteen base pair An alternative form of base pairing in which the purines rotate from the anti to the syn

confi guration and form base pairs as follows: A( syn ) C6(NH 2 ), N7 with T ( anti ) C4(O), N3(H); G ( syn ) C6(O), N7 with C ( anti) C4(NH 2 ), N3(H) .

Indel In sertion or Del etion of a small number of nucleotides in the DNA structure. Insertional mutagenesis Mutation by insertion of one or more nucleotides. Often used to denote inactivation of the genes

by insertion of large transposable elements. Inversion A rearrangement of the chromosome so that the order of the nucleotides is reversed: if the

normal order is ABCDEF, the order AEDCBF would constitute an inversion. L1 element A common retrotransposon found in the human genome. Missense mutation A change in a gene which results in a change in the meaning of a codon: e.g. the change from

GAA (glutamic acid) to GUA (valine). Nonsense mutation A mutation which results in one of the termination codons UAA, UAG or UGA. Point mutation A mutation involving one or a few nucleotides as distinguished from insertions, deletions and

duplications involving hundreds, thousands or more nucleotides. Proofreading In DNA synthesis the process where an exonuclease checks a newly inserted nucleotide for

goodness of fi t. Sometimes referred to as editing. Recombination Processes which result in splicing together of portions of different DNA molecules. Retrotransposon A transposable element which can shift its position in DNA via an RNA intermediate. Slippage In DNA replication, the addition or deletion of nucleotides as a result of movement of repeated

bases in either the template or newly synthesized strand during synthesis resulting in either repeated replication of the same segment or loss of one or more of the repeats when slippage occurs in the template.

Synonymous/silent mutation A nucleotide change that does not change the meaning of a codon, e.g. the change from GGU (glycine) to GGA (glycine).

Termination codon A triplet which does not specify a particular amino acid and is used to signal the termination of a peptide chain. UGA, UAA, and UAG are termination (ter) codons.

Translocation Attachment of a segment of one chromosome to a different (non-homologous) chromosome. Transposition The movement of a transposable element from one position in the genome to another. Transition The mutational change from a purine to another purine or a pyrimidine to another pyrimidine.

G ← → A and C ← → T are the possible transitions. Transposon A DNA sequence able to move from one position to another within the genome. Movements are

generally rare and are catalyzed by special enzymes coded for by the transposon. Transversion The mutational change from a purine to a pyrimidine or a pyrimidine to a purine. A ← → T,

G ← → T, C ← → G, C ← → A are possible transversions. Ubiquitin A conserved small (76 amino acids in human) protein which, when covalently added to proteins

in single or multiple copies, serves as a signal for processes such as degradation and/or changes in conformation.

Unequal crossing over Recombination between homologous chromosomes that are not precisely paired resulting in an unequal exchange of material. Usually occurs in regions of duplicated genetic material.

Watson – Crick base pair Base pairs found in the double helical structure of DNA. G and C pairs both in the anti confi guration are held together by hydrogen bonding between C2(NH 2 ), N1 (H) and C6(O) of guanine and C2(O), N3 and C4(NH 2 ) of cytosine. A and T pairs are held together by hydrogen bonding between N1 and C6(NH 2 ) of A and N3 and C4(O) of T .



mutagenic activity, was modifi ed to account for such acti-vation by the incorporation of a liver extract to media on which presumptive mutagens were tested [9] .

We live in an environment that is essentially 55.6 Molar water. Given the law of mass action, hydrolytic reactions are inevitable. It has been estimated that we lose about 18,000 bases in every 24-hour period as a result of hydroly-sis of the glycosidic bond [1] . The abasic sites so created are intrinsically mutagenic and organisms have devised a set of enzymes to survey and repair the damage. However, the most reactive mutagen in our environment is undoubt-edly oxygen. Breathing, however unavoidable, is inherently dangerous! The electron transport chain by which ATP is generated results in the generation of reactive oxygen spe-cies (ROS) that produce the hydroxyl radical OH. When formed in proximity to DNA this species produces a vari-ety of oxidation products, of which a guanine with a satu-rated imidazole ring (8-oxoguanine) is the most important. It is so important that enzymes utilizing different tactics are produced to overcome the effects [10] . The fi rst, OGG1, is a glycosylase that removes the damaged base and sets off the base excision repair sequence. 8-oxoguanine in a DNA template strand has an increased probability of pairing with adenine resulting in G:C → T:A transversions. The human enzyme MYH (bacterial mutY homolog) removes As inserted opposite such damaged Gs. Base excision repair then occurs. Most of the time, the correct C will be inserted, but if not, the cycle can be repeated until an apoptic reac-tion ensues.

Some mutagenic changes, particularly those close to repeated sequences, seem to defy the specifi city rules. Studies of the infl uence of the surrounding sequence on mutation permitted the deduction, supported by evidence with in vitro models, that what at fi rst appeared to be base substitution mutagenesis might actually be the result of slip-page errors in DNA replication [11] . Frameshifts (indels) are also likely to occur at regions of repeated sequence and the models account for such changes as a result of misalign-ments of either template or newly synthesized strand as a result of “ slippage ” during replication. Dissociation and reassociation of DNA strands occurs repeatedly and when there are repeated sequences, the reassociation may occur so that some of the repeated sequences in the newly synthe-sized strand “ loop out ” of the structure making addition of extra bases possible.

SPECIFICITY RULES

Most (early) considerations of mutational specifi city and frequency focused on considerations of hydrogen bond-ing and on the base pairs suggested by Watson and Crick. Occasionally, an alternative pairing, the Hoogsteen base pair, was suggested to account for particular cases of

specifi city [12] . Notwithstanding the ability of such mod-els to account for the data, it is not at all clear that hydro-gen bonding is critical for successful DNA synthesis [13] . A non-polar thymine analog, (2,4 difl uorotoluene) unable to form hydrogen-bonded base pairs can be incorporated selectively opposite adenine or an equivalent non-polar analog. The results obtained with such non-polar analogs depend on the polymerase used for the study. For some enzymes shape of the substrate is the determining factor; for others hydrogen bonding remains an important feature. Such studies with different polymerases indicate that the chemical nature of the substrate nucleotide is not the sole determinant of mutagenic specifi city.

A favorite subject for study has been an abasic site, a point in the sequence at which a base has been removed without breaking the backbone DNA chain. In the absence of a base to serve as template, one might suppose that if replication were to proceed it would do so with bases added at random. In fact, this is not how it works [14] . Although the abasic site is a barrier to normal synthesis, some polymerases are able to insert bases opposite such sites and to extend DNA chains past the lesion. Bacterial replicative enzymes tend to insert As opposite abasic sites and there are structural studies to indicate why this should be the preferred result. Yeast will insert a C or an A. Yeast, it turns out, contains a special protein (rev1), essentially a nucleotidyl transferase, that inserts Cs at the end of grow-ing chains and will insert a C opposite an abasic site. Mammalian cells are likely to insert Gs or As. The muta-genic specifi city in different organisms turns out to be due to the specifi cities of their polymerases. The surprising fi nding, particularly in view of the emphasis on base pair-ing in the 1980s, is that both the frequency and specifi city of the errors are determined by the particular polymerase used for replication [15, 16] .

There are some rules that apply to most of the polymer-ases. Particular mutagens do react with DNA to produce products with unique pairing properties that predict the type of mutation to be obtained. The important guanine modifi -cations have an increased propensity to pair with adenine, leading to G:C → T:A transversions. Guanine also reacts with many of the metabolically activated polycyclic hydro-carbon derivatives implicated as carcinogens and the result-ing products when replicated also appear to give primarily G → T transversions followed by G → C transversions. In contrast, most “ spontaneous ” mutations at sites of G are transitions, explained as the conversion by deamination of methylC in G(methyl)C pairs to G:T which replicates to give a G(methyl)C → A:T transition. Survey of genes for the types of mutation observed, so-called mutational spectra, can give clues as to the origin of the mutations observed. So, for example, the prevalence of G → T mutations in lung cancers is taken as evidence for the role of tobacco related polyaromatic hydrocarbons and nitrosoamines as mutagens [17] . Unfortunately, such mutagenic spectra cannot identify



the precise lesion or agent involved. In the case just cited either oxidation of guanine or the addition of a poly aromatic hydrocarbon group lead to the same mutational change.

MUTAGENIC POLYMERASES

The conclusion that organisms evolve their own mutation rate and, by providing variation, their own rate of evolution comes from the discovery of mutations in bacteria and in yeast that make the organisms “ mutagen stable ” . For exam-ple, treatment with ultraviolet light will kill such mutant organisms, but the mutation frequency will not increase among the survivors. An interpretation of the fi nding is that mutation requires that an altered DNA be replicated and that during replication past the lesion produced by the mutagen, an error occurs. If the DNA lesion induced by the treatment ordinarily blocks replication, then what is termed “ trans lesion synthesis ” (TLS) is required in order for muta-tion to occur. The fi nding of “ mutagen stable ” mutants indi-cated that proteins different from those used for replication were required for TLS. In the 1990s it was discovered that Escherichia coli , yeast, humans and other organisms code for a set of DNA polymerases distinct from the replicative polymerases and with specifi cities inherent in their struc-ture. One polymerase, polymerase eta (the polymerases are assigned Greek letter names) is adept at bypassing pyrimi-dine dimers produced by ultraviolet radiation. Such dimers are major products of exposure to sunlight and polymerase eta synthesizes past the T^T lesion without error, inserting two As. A defi ciency in this enzyme results, in humans, in the variant form of xeroderma pigmentosum, a disease characterized by the induction of numerous skin tumors. In the absence of polymerase eta, a second enzyme, prob-ably polymerase iota takes over, but this enzyme does make errors (mutations) when synthesizing past pyrimidine dim-ers. There are about 16 different human DNA polymerases known ( Table 1.2 ) and these enzymes have been classi-fi ed into different groups based on structural homologies [15, 16] . Polymerase eta is a member of the Y family of polymerases which includes Pol ι (iota), Pol κ (kappa), and Rev1 (terminal deoxycytidyl transferase). Polymerase zeta is a member of the B family of polymerases that also includes the major replicative polymerases. The Y family of polymerases is characterized by a relaxed specifi city. Structural studies show that the pockets, which in replicative polymerases fi t tightly around the incoming nucleotides, are relaxed permitting “ wrong ” bases to be incorporated. The enzyme REV-1 pairs an incoming C with an arginine in the protein sequence rather than with the ostensible templating base [18] ! On occasion, the incorporation of incorrect bases is apparently preferred by mammalian cells as a survival mechanism, since blocked replication forks lead to lethal double strand breaks in DNA.

One characteristic of replicative DNA polymerases in vivo is their processive nature when part of the replicative complex. That is, the polymerase remains attached to the DNA growing point after incorporation of a nucleotide, poised for the addition of the next base. Damaged nucleo-tides in the template block this progression. In order for the Y family polymerases to promote TLS, they must in some way displace the replicative polymerase from the growing point. This is accomplished via the sliding clamp, PCNA (proliferating cell nuclear antigen) which encircles the DNA at the replication fork [19] . Addition of ubiquitin to the clamp serves as a signal for combination of the Y fam-ily polymerases which can then rotate into the active site as the replicative polymerase falls off [20] . These error-prone polymerases are not processive and after addition of a few nucleotides they fall off. The details of polymerase action at growing points containing damaged nucleotides are an object of active research at the present. Its practical sig-nifi cance lies in the possibility of interrupting the action of such auxiliary polymerases, thereby diminishing the overall mutation rate.

MUTATION MODIFIERS

In spite of the explanations given in many elementary texts, the free energy differences between correct and incorrect base pairs are very small, at most 0.4 kcal/mol. This means that in a water solution, in which there is much competitive hydrogen bonding, a correct base pair is only about twice as likely to form as is a mismatch. The major contribu-tion to specifi city is clearly the replicative polymerases: the structural nature of the pockets into which incoming nucleotides fi t and the kinetic interactions between elon-gation of the chain and reversal of the reaction accounting for this specifi city. The free energy differences between correct and mismatched bases for a reaction catalyzed by a replicative polymerase ( Drosophila melanogaster polymer-ase alpha) indicated a difference of 4.9 kcal/mol, equiva-lent to a discrimination factor of about 1 in 3000 [21] . The in vitro measured error frequency of the different polymer-ases ( Table 1.2 ) varies from a low of about 1 in 100,000 for the different B family replicative polymerases to more than 3.5 per 100 for human polymerase eta [15] . However, the mutation rate in organisms is likely to be between 10 � 8 and 10 � 9 per nucleotide per generation [22, 23] , a value four to fi ve orders of magnitude lower. Organisms attain this high level of fi delity by two major processes contributing to specifi city consequent to replication: proofreading and mismatch repair (MMR).

DNA synthesis can be considered as a series of steps in which the growing chain is elongated and then the newly inserted base is checked, or proofread, to determine if it meets built in pairing specifi cations ( Fig. 1.1 ). Terminal



TABLE 1.2 Human DNA polymerases

Polymerase Family Exo activity

Error rate � 10 � 5

Comments (suggested function) Reference Single base � 1 base Indel

Pol G ( γ ) A � 4.5 1.7 Mitochondrial replication JBC 276:38555(2001) PolQ ( Θ ) A � 240 140 TLS, SHM NAR 36: 3847 (2008) PolN ( ν ) A � 350 17 T opposite template G Bypass

thymine glycols DNA Repair 6: 213 (2007)

PolA ( α ) B � 5 3.1 Replication Biochemistry 30: 11751 Pol D ( δ ) B � 0.425 1.79 (calf thymus) replication Biochemistry 30: 11751 Pol E ( � ) B � 1.1 0.5 (calf thymus) replication Biochemistry 30: 11751 (1991) Pol Z ( ζ ) B � 130 4.4 (yeast) extends mismatch Cell Res 18:174 (2008) NAR 34: 4731 (2006) Pol B ( ß ) X � 18 1.5 Gap fi lling, BER JBC 274: 3642 (1999) Pol L ( λ ) X � 90 450 NHEJ, BER, Meiosis JBC 278: 34685 (2003) Pol M ( μ ) X � 150 42,000 NHEJ Mol. Cell. Biol. 21:7995 (2001) TdT X � Non-templated Terminal

transferase

Pol H ( η ) Y � 3500 240 SHM JMB 312:335 (2001) Pol I (i) Y � 72,000 G opposite template T No

known function, BER? Science 291:2156 (2001)

Pol K ( κ ) Y � 500. 180. JBC 275:39678 (2000) REV1 Y � 3000 (CvsA) Error rate of 12,000 for C

opposite Abasic sites FEBS Lett. 520:88 (2002)

hTERT RT � 200 Telomerase reverse transcriptase Nuc. Ac. Symp. Ser 333: 137 (1995)

TLS, trans lesion synthesis; BER, base excision repair; SHM, somatic hypermutation, NHEJ, non-homologous end joining; hTERT, human telomerase terminal transferase; JBC, Journal of Biological Chemistry; NAR, Nucleic Acids Research; JMB, Journal of Molecular Biology.

BINDINGCONFORMATIONAL CHANGE PCNA UBIQUITINATION POLYMERASE SWITCHING

ADDITION

PROOFREADING

RECOGNITION PCNA UBIQUITINATION EXONUCLEOLYTIC ACTION EXTENSION

POLYMERASE SWITCHING

ELONGATION

MUT S SEARCHING MUT L RECRUITMENT

MISMATCH REPAIR

FIGURE 1.1 Schematic outline of the competition between extension and proofreading in DNA synthesis.



bases that do not fi t are removed by exonucleolytic action. The requisite 3 � → 5 � nuclease activity is either built into the structure of B class polymerases or exists as a separate but closely associated protein(s). Y family polymerase mem-bers are devoid of exonuclease activity. Exonucleolytic proofreading results in about a hundred-fold increase in the fi delity of replication. Mutations in either the exonucle-ase or exonuclease domains have mutator properties, pro-ducing additional mutations in every round of replication. Organisms can “ fi ne tune ” their proofreading, and mutants (of bacterial viruses) have been isolated in which the rate of spontaneous mutation is lowered because of an increase in the effi ciency of proofreading. Such an increase comes at a cost in energy since the ATPs required to provide the pyrophosphates required for polymerization are wasted. Even replication events with normal bases involve proof-reading. About 6 to 13% of the polymerization events result in an excised (proofread) base [24] . The replication process can be depicted as a competition between proofreading and further elongation [14] since once the chain has been elon-gated fi ve or six nucleotides beyond a mismatch it appears immune to proofreading. The elongation step is distinct from the initial addition opposite any particular base ( Fig. 1.1 ). Some of the enzymes of the Y series are relatively effi -cient in the addition of a nucleotide opposite a non-pairing template but are unable to elongate the resulting product. It has been suggested that polymerase zeta, a B family polymerase, has as a function the elongation of mismatched bases inserted by iota and other error prone polymer-ases [16, 25] . The events in trans lesion synthesis can be described as follows: the replicative complex recognizes a mismatch or altered base. This triggers ubiquitination of the PCNA clamp which in turn results in dissociation of the replicative complex from the DNA and allows access of a Y family polymerase to the growing point [20] . A deoxy-nucleotide is added, and before the replicative complex and its associated proofreading activity can access the mis-match, polymerase zeta replaces the Y family polymerase and elongates for a few nucleotides. Polymerase zeta then falls off and is replaced by the normal replication complex. Ubiquitination of PCNA plays a critical regulatory role in the process [20] . A variation of this scenario suggests that replication proceeds asynchronously until the next initia-tion sequence, leaving a gap which is subsequently “ fi lled in ” by a process similar to that described. Superimposed on these events must be the availability of the different deoxy-nucleotides used for synthesis. Alterations in the pool size of the different DNA constituents can affect the selection of bases and altering relative pool sizes can be mutagenic [26] . Whatever the sequence of events, the result of any particular elongation attempt will be determined by the various com-petitions for access to the nucleotide at the growing point. The result of any single specifi c replication event cannot be predicted.

MISMATCH REPAIR

Newly synthesized DNA in humans and other organisms is subject to yet another inspection by the set of proteins con-stituting the mismatch repair (MMR) system. These proteins detect mismatches in the DNA: both base pair mismatches and mismatches due to small additions or deletions. In bac-teria, in which the process was studied fi rst, the detection is carried out by a single protein acting as a homodimer, the mutS protein, which when bound to the mismatch recruits a second protein dimer, mutL. In the enteric bacteria this ATP dependent complex activates the endonuclease activity of a third protein, mutH, which makes a single stranded break in the error-containing strand. The nicked strand is unwound by a helicase encoded by the UvrD gene and the displaced strand is degraded by an exonuclease. The resulting sin-gle stranded gap is then fi lled by the replicative polymer-ase. The key to the successful operation of this scheme is making sure that the newly synthesized strand including the “ error ” is the one removed. In Escherichia coli this trick is accomplished by a special methylation mechanism. Adenines at GATC sites are methylated on both strands but the methylation of the newly inserted adenine is accom-plished only after replication. Immediately after replica-tion the newly synthesized strand is unmethylated. It is this hemi-methylated DNA which is the substrate for mismatch repair and it is the unmethylated, i.e. newly synthesized, strand which is removed [1] .

Eukaryotic cells have a more complex, although clearly similar, mismatch repair mechanism [27] . Instead of a single mutS protein, eukaryotes have fi ve, three of which (MSH2 [MutS homolog], MSH3 and MSH6) form dimers with slightly different specifi cities. There are four MutL homo-logues (MLH1, MLH2, PMS1 [post meiotic segregation protein]), and PMS2 which also function as heterodimers. The MSH2:MSH6 heterodimer recognizes base – base mis-matches and small insertions or deletions, the MSH2:MSH3 complex specializes in recognition of larger insertions and deletions. As the names indicate, certain of these proteins also play an important role in meiosis. There is no MutH analog. The adenine methylation recognition mechanism appears confi ned to enteric bacteria. In vitro reconstructions of the eukaryotic mismatch repair system use a free 3 � OH end (i.e. a nick in the DNA) to identify the newly synthe-sized strand and it appears likely that in vivo it is the grow-ing point of the DNA (or an unligated Okazaki fragment) that provides the MMR signal. Eukaryotic MMR is more closely tied to replication as compared to the enteric bacte-ria. The MSH proteins have been shown to bind to PCNA which locates them at the site of the DNA growing fork [19] .

Organisms defi cient in their ability to make one of the mismatch repair proteins have increased mutation rates. The medical interest in MMR dates from the discovery that an inherited colon carcinoma syndrome can be traced



to a defi ciency in the MMR proteins [28, 29] . The most frequent culprits are the hMLH1 (human mutL homolog) and hMSH2 genes, followed by hPMS2 and hMSH6. Analyses of tumor tissue show that the promoters of these MMR genes are frequent targets of epigenetic inactivation by methylation [30] . The absence of a functional MMR system is often signaled by an increase in microsatellite instability. The “ microsatellites ” are regions of mono-, or di-nucleotide repeats (e.g. CACACACACA) that are poly-morphic, i.e. in which the actual number of repeat units at a particular location differs among individuals. The number of repeat units at each locus is inherited and is the basis of much DNA “ fi ngerprinting ” . “ Instability ” is observed as a detectable increase in the number of such repeats, easily demonstrated by gel electrophoresis. Individuals defi cient in MMR may have thousands of microsatellite instabili-ties throughout the genome but a panel of fi ve selected loci is generally used for testing. Instability at two loci serves as a positive signal [31] . Bound MMR proteins may also serve as a signal for apoptosis. Organisms defi cient in the O 6 -methylguanine methyltransferase protein are exquisitely sensitive to killing by methylating agents. The cells become much less sensitive when made MMR defective, possi-bly because of a loss of a signal from the MMR proteins combined at the O 6 methylG:T mismatch. The mechanism is important because MMR defi cient cells with mutagenic lesions that should be signals for apoptosis, survive, repro-duce and propagate mutations [32] .

One of the unexpected discoveries of the 1990s was the fi nding that about 30 mostly neurodegenerative diseases, including Fragile X syndrome, Huntington chorea, myo-tonic dystrophy and Friedrich’s ataxia are due to expansions of simple repeats in the genome [33] . For example, a CAG sequence which occurs from 6 to about 35 times in nor-mal individuals expands to up to 100 repeats in individu-als affected with Huntington ’ s chorea. In Friedrich’s ataxia, a normal GAA sequence occurring from 7 to 22 times in an intron may expand to 200 to 1700 units. The origin of these mutations is mysterious. Model systems have been developed in which the infl uence of slippage during repli-cation has been studied. It seems that there is a role for the mismatch repair system [34] but exactly what sets off these changes is a mystery.

MUTATION OUTSIDE THE REPLICATION CYCLE

Although it is simplest to consider mutation as a conse-quence of DNA replication, DNA turnover is not limited to the S phase. Insofar as DNA repair processes involve excision and reconstitution of the excised region, there is a chance for mutation during the repair process, particularly if an error prone polymerase is recruited to fi ll in the gap. Such

an error will create a mismatch, but correction of such mis-matches during interphase might occur using either strand as a template and resulting in “ fi xation ” of such mutations. This sequence seems to account for the phenomenon of somatic hypermutation during the immune response: per-haps the only instance in which mutations are an essen-tial part of a normal biological process [35] . The fi rst step, which depends on transcription of the immunoglobin gene, involves activation of a single strand specifi c cytidine deam-inase (activation-induced cytidine deaminase, AID). This enzyme converts cytidine to uracil creating a U:G mismatch. The mismatch is recognized either by the MMR pathway or by uracil glycosylase. During the ensuing repair processes, involving polymerase eta and the rev1 cytidine terminal transferase protein, mutations are generated. It has not been determined whether somatic hypermutation occurs during the S phase, but there is no reason why it should be limited to this period. Somatic hypermutation is limited to portions of the immunoglobulin gene, in particular cells at only limi-ted stages of their differentiation. It is not clear what gives the process its specifi city. Its interest lies not only in the importance of the immune phenomenon itself, but in the possibility that under particular circumstances something similar might occur, say, in tumorigenesis. This possibility is highlighted by the discovery of somatic hypermutations in the BCL6 and CD95 genes [36] .

SPONTANEOUS MUTATION AND TUMORIGENESIS

Mutations are clearly important in tumorigenesis and it is clear that natural selection, in the sense of selection of the clones fi ttest for reproduction in the host’s environ-ment, plays a major role. A fi rst question is whether tumors are hypermutable or whether the accumulation of muta-tions in tumors can be accounted for solely by selection. Hypermutability is an old suggestion put in modern terms in a series of papers by L. Loeb [37] . A parallel series of papers, summarized by W. Bodmer [38] , argues that the spontaneous human mutation rate is suffi cient to account for the mutations observed. Mismatch repair defi ciency and its associated increased mutation rate is certainly associated with a sub-class of colon carcinomas. However, such defi -ciency accounts for only about 15% of colon carcinomas and the tumors have properties (e.g. a relatively stable kary-otype) that distinguish them from other solid tumors. It is also (now) clear that tumor DNA is separated from normal tissue by many mutations. When analyzed by methods that sequence DNA derived from tumors and neighboring sur-rounding tissue, the tumors show many more mutations than the surrounding normal tissue. In one study in which 274 megabases of tumor DNA was screened for point mutations in exons of protein kinase genes the investigators detected



1007 mutations: 921 single base changes, 54 nonsense muta-tions and 219 silent mutations [39] . Assuming an average nucleotide size of 1500 bases per gene this fi nding implies a mutation frequency of about 0.5% (0.005), or about one new mutation for every 200 genes. One of the obser-vations in this, and similar studies, is that no two tumors have precisely the same pattern of mutation – there is little overlap in the mutations observed although some mutations, e.g. in TP53, do tend to recur. Since we expect that each of us inherits about three new mutations in 50,000 genes (two sets of 25,000) this seems to show that the tumors must be highly mutable. The adherents of the “ selection is suffi cient ” school argue that this need not be so. Direct measurements of the somatic mutation rate in human cells are rare but a recent measurement gives a value of 1.06 � 10 � 6 muta-tions per cell division for the PIG-A gene [22] . There are 249 amino acids in this gene coded for by 747 nucleotides, which gives a mutation rate of 1.4 � 10 � 9 per nucleotide per generation. (For comparison, an estimate of the aver-age mutation rate for 20 genes involved in Mendelian dis-ease is 1.8 � 10 � 8 per nucleotide per generation [23] .) The adult human has approximately 5 � 10 13 to 10 14 cells (this number is at best a guess by pathologists). Since the number of divisions required to produce a population of N cells is N – 1; this means that there are 1.41 � 10 � 9 � 2 � 3 � 10 9 nucleotides (the number of nucleotides in a diploid genome) � 10 14 divisions or 8.5 � 10 14 new mutations pro-duced during development. Only about 5% or less of these will be in the genes, but even so this means 4 � 10 13 muta-tions distributed throughout 25,000 genes. This is about equal to the number of cells, so that most cells will have at least one new mutation in their genes. Based on the Poisson distribution, many cells will have no mutations but others will have two, three, four or more. If, as seems to be indi-cated by recent whole genome association studies, the “ non-genic ” region of the genome is not as devoid of information as we suppose, the number of effective mutations will be even higher. Even in the absence of a mutator activity the selectionists argue that there will be suffi cient genetic vari-ation to drive tumor progression. The experimental obser-vations are that mutations in genes affecting DNA repair, and therefore mutation rate, are, as a class, frequently found mutated in tumors. The debate has practical consequences. The common observation that cancers arise in the elderly is often interpreted as indicating that there needs to be an accumulation of individual (mutational) changes before an overt tumor develops. Many estimates suggest that fi ve or six changes need to accumulate. Even a slight decrease in mutation rate would raise the age of incidence to greater than the average life span. If cancers have a mutator phe-notype, it is argued that it may be easier to fi nd drugs to counteract this phenotype and thus increase the age of fi rst incidence to greater than the average life span!

The frequent occurrence of new mutations in all somatic cells makes it important to distinguish between mutations

that are important in the etiology of a condition being inves-tigated and those present by accident. For example, con-sider two cells spatially separated in a tissue. Both will have accumulated a different complement of neutral mutations during their separate development. Collection of the tissue and sequencing by standard methods will not reveal any of these new mutations because sequences that represent less than about 10% of the genome are dismissed as noise in standard sequencing. Suppose now that one of these cells develops a new mutation that leads to proliferation. Cells of this new clone will all contain the new mutation but in addition, they will contain all of the mutations that have occurred in that particular cell during its develop-ment. Sequencing of a tumor derived from that clone will reveal not only the mutation responsible for the selection (the “ driver ” ), but also all of the others (the “ passengers ” or “ hitchhikers ” ) that have accumulated previous to the transformation.

The evidence for this theoretical formulation comes from an examination of the types of mutation observed. If mutation is random and if there is no selection, then about 25 to 30% of all mutations, depending on the codon usage of the gene being studied, should be silent or synonymous, resulting in no amino acid change. Demonstrating that the proportion of silent mutations is what would be expected for random mutation is a sign that such mutations have not been selected. In the data set referred to above [17] , 219 out of 1007 mutations or about 20% were silent, close to, but slightly below the expected value for pure random muta-tion. The investigators conclude that some of the mutations are indeed “ drivers ” and functionally signifi cant, but it takes both sophisticated statistics and biological insight to deter-mine which [40] .

THE ROLE OF DNA STRUCTURE

Although the Human Genome Project was offi cially com-pleted in 2003, a 2008 paper [41] concludes that what was sequenced may actually represent only a minor allele! Continued advances in techniques for the analysis of DNA coupled with analysis of the genomes of a wider range of individuals confi rms the fi ndings that not only are there large numbers of single nucleotide polymorphisms (SNPs) but that structural variations including insertions, deletions and inversions of the DNA sequence may involve more base pairs than are found in the SNPs. Changes in copy number have been associated with both Mendelian and complex human traits [42] . The repeated elements in our genome promote instability by at least two major mechanisms, transposition and unequal crossing over, and it may be that these mechanisms are at least as likely to be responsible for mutagenic change (as defi ned phenotypically) as are any of the point mutational mechanisms. One of the startling



revelations of the year is the observation that a group of identical twins display mosaicism in the copy number of their genes [43] . Copy number variation was observed in twins both concordant and discordant for particular traits. A deletion associated with MLL was found in one twin pre-viously diagnosed with the disease. The genetic identity of monozygotic twins has been a given in genetic research for years. The report suggests that recombination events can occur during mitotic development of individuals and that such variations may have medical relevance.

Aberrant recombination of repeated elements in the genome can result in duplication, deletion or inversion of large regions of the genome ( Fig. 1.2 ). Insertional mutagen-esis due to the introduction of a transposed element into a gene can result in disruption of the gene product. The most prevalent transposable elements are the SINES (short inter-spersed nuclear elements) and the LINES (long interspersed elements). Full sized LINE elements are about 6.1 kb in size and contain coding sequences for gene products essen-tial for their transposition within the genome. Humans may have 200,000 to 500,000 of these elements in their genome, of which the most frequent is the L1 (LINE-1). Only a few

such elements retain their capacity to catalyze movement [44] . The others have suffered a variety of sequence changes in the course of history resulting in their inactivation. The SINE elements, of which the most prevalent are the Alu sequences of about 300 bases, may be present in approxi-mately 1,000,000 copies throughout the human genome. These elements cannot catalyze their own movement but can apparently transpose by utilizing some of the enzy-matic machinery produced by LINE elements. Transposons are recognizable by the target site duplications (TSD) that are found at either end of the insertion sites. Such duplica-tions of about 2 to 10 bases occur as a result of the insertion mechanism, which at one point involves making a staggered nick in the double helix somewhat similar to the staggered cuts made by restriction enzymes, although there seems to be no specifi c sequence recognition for the insertion sites.

Well over 30 pathological conditions have been associ-ated with new insertions of Alu sequences and about 11 with insertion of L1 elements [45] . Eight of the L1 elements are reported on the X chromosome and 9 of the Alu sequence are X-linked. This unexplained excess on the X does not seem to be accounted for by the bias derived from the necessary

a. DUPLICATIONS: Unequal Crossing Over B’B CA

A B C D a

A B C D A/a D C B A/aA B

a

A B B/b b’ c

a cb’b a b/B’ C

(homology ; crossover )

b. DELETION A

a A/a

B a / A D C

C D C B

c. INVERSION

C

D

FIGURE 1.2 Cartoon showing how repeated elements in the genome can lead to chromosomal aberrations. (a) Duplication due to unequal crossing over. During meiosis recombination of homologous chromosomes containing displaced repeated elements can result in one recombinant obtaining an extra copy of the repeated element and the second chromosome losing a copy. (b) Deletion due to two trans-posable (repeated) elements inserted in the same chromosome in the same orientation. Pairing of the homologous regions of the duplicated transposable elements followed by recombination results in a daughter chromosome in which the genetic material between the chromo-somes has been deleted. The loop including a copy of the transposon and of the genes intervening is presumed to be lost in replication. Recombination between non-homologous chromosomes containing such repeated sequences can lead to translocations. (c) Inversion due to two transposable elements inserted into the same chromosome in opposite orientations. As shown in the diagram, recombination will result in inversion of the chromosome order.



dominance of all mutations in males. It has been estimated that there is a new Alu transposition about once in every 20 births and that the ratio of disease producing transpositions as compared to single nucleotide changes is 1 in 2000.

The actual process of retrotransposition is estimated to account for only about 15% of the events leading to struc-tural variation, possibly because most of the elements are no longer active [44] . Most of the structural aberrations occur as a result of recombination events between repeated sequences [41] . Unequal crossing over ( Fig. 1.2 ) can produce both addition and deletion of particular sequences. The production of deletions and inversions depends on whether the repeated sequences occur in the same or inverted orientation ( Fig. 1.2b,c ). Recombination between repeats in different chromosomes can result in transloca-tions. Our current understanding of the structure of the genome therefore indicates it to include numerous sources of instability. Most such changes will be eliminated when they occur at meiosis because they result in abnormal devel-opment. Changes occurring in somatic cells may result in pathological change and we are only beginning to under-stand such instability. The numerous repeats and retroviral like elements in our genome act as potential mutagens.

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CHAPTER 1 Mechanisms of Mutation - Elsevier · CHAPTER 1 Mechanisms of Mutation 5 mutagenic characteristics of such compounds. A major development was the discovery that metabolic