7/28/2019 Mutation 4th
1/40
MUTATION
7/28/2019 Mutation 4th
2/40
7/28/2019 Mutation 4th
3/40
Introduction
Mutations can happen at any time and in any cell
The phenotypic effects can range from minoralterations that are detectable only bybiochemical methods to drastic changes in
essential processes that cause, at one extreme,unrestrained cell proliferation (cancer) or, at theother extreme, the death of the cell or organism
The effect of a mutation is determined by the typeof cell containing the mutant allele, by the stage inthe life cycle ordevelopment of the organism thatthe mutation affects, and, in diploid organisms, bythe dominance or recessiveness of the mutant
allele
7/28/2019 Mutation 4th
4/40
Definition
A mutation is a change in a DNA base-pair(substitution, deletion, or insertion of a basepair) or a chromosome (deletion, insertion,
or rearrangement)a.Somatic mutations affect only the individual in
which they arise
b.Germ-line mutations alter gametes (tissues that
produces eggs & sperm) and passed the next
generation
7/28/2019 Mutation 4th
5/40
What can happen when a mutation occurs in the DNA
(e.g. sickle cell anemia)
Concept of a mutation in the protein-coding region of a gene. (Note that
not all mutations lead to altered proteins and that not all mutations are in
protein-coding regions)
7/28/2019 Mutation 4th
6/40
Mutations are quantified in two different ways:
a. Mutation rate is the probability of a particular
kind of mutation as a function of time (e.g.,
number per gene per generation) or
# mutation per nucleotide pair or gene pergeneration
b. Mutation frequency is number of times a
particular mutation occurs in proportion to the
number of cells or individuals in a population(e.g., number per 100,000 organisms) or
# of a particular mutation per 100,000
organisms
7/28/2019 Mutation 4th
7/40
Types of mutations
Two types of point mutations:1. Base pair substitutions
a. Transitions
Convert a purine-pyrimidine to the other purine-
pyrimidine (e.g., AT to GC or TA to CG)
4 types of transitions; A G and T C
Most transitions results in synonymous substitution
b. Transversions
Convert a purine-pyrimidine to a pyrimidine-purine (e.g.,
AT to TA, or AT to CG)
8 types of transversions; A T, G C, A C, and G
T
Transversion more likely to result in nonsyn substitution
7/28/2019 Mutation 4th
8/40
7/28/2019 Mutation 4th
9/40
2. Base pair deletions and insertions
can change the reading frame of the mRNA
downstream of the mutation, resulting in a
frameshift mutation.
a. When the reading frame is shifted, incorrect amino
acids are usually incorporated.
b. Frameshifts may bring stop codons into the reading
frame, creating a shortened protein.
c. Frameshifts may also result in read-through of stop
codons, resulting in a longer protein.
d. Frameshift mutations result from insertions ordeletions when the number of affected base pairs is
not divisible by three.
7/28/2019 Mutation 4th
10/40
Types of base pair substitutions and mutations
7/28/2019 Mutation 4th
11/40
Types of base pair substitutions and mutations
7/28/2019 Mutation 4th
12/40
Effect of a nonsense mutation on translation
7/28/2019 Mutation 4th
13/40
Types of mutations in ORFs:
Nonsynonymous/missense mutation
Base pair substitution results in substitution of a different amino
acid.
Nonsense mutation
Base pair substitution results in a stop codon (and shorter
polypeptide).
Neutral nonsynonymous mutationBase pair substitution results in substitution of an amino acid
with similar chemical properties (protein function is not altered).
Synonymous/silent mutation
Base pair substitution results in the same amino acid.
Frameshift mutations:
Deletions or insertions (not divisible by 3) result in translation of
incorrect amino acids, stops codons (shorter polypeptides),or
read-through of stop codons (longer polypeptides).
7/28/2019 Mutation 4th
14/40
The nine codons that can result from a single base change in the tyrosine codon UAU. Blue
arrows indicate transversions, gray arrows, transitions. Tyrosine codons are in boxes. Two
possible stop (''nonsense") codons are shown in red. Altogether, the codon UAU allows for six
possible missense mutations, two possible nonsense mutations, and one silent mutation
7/28/2019 Mutation 4th
15/40
Reverse Mutations and Suppressor
Mutations
1. Point mutations are divided into two classes based on their effect on
phenotype:
a. Forward mutations change the genotype from wild type to mutant.
b. Reverse mutations (reversions or back mutations) change the genotype from mutant
to wild-type or partially wild-type.
i. A reversion to the wild-type amino acid in the affected protein is a true reversion.ii. A reversion to some other amino acid that fully or partly restores protein function
is a partial reversion.
2. Suppressor mutations occur at sites different from the original mutation, and
mask or compensate for the initial mutation without actually reversing it.
Suppressor mutations have different mechanisms depending on the site at
which they occur.
a. Intragenic suppressors occur within the same gene as the original mutation, but at a
different site. Two different types occur:
i. A different nucleotide is altered in the same codon as the original mutation.
ii. A nucleotide in a different codon is altered (e.g., an insertion frameshift is
suppressed by a nearby deletion event).
7/28/2019 Mutation 4th
16/40
b. Intergenic suppressors occur in a different gene (the suppressor gene)from the original mutation. Many work by changing mRNA translation.
i. Each suppressor gene works on only one type of nonsense, missense or
frameshift mutation.ii. A given suppressor gene suppresses all mutations for which it is specific.
iii. Suppressor genes often encode tRNAs that recognize stop codons and insert anamino acid, preventing premature termination of translation.
(1) Full or partial function of the polypeptide may be restored.
(2) The effect depends on how compatible the substituted amino acid is with protein function.
iv. Nonsense suppressors fall into three classes, one for each stop codon (UAG,
UAA and UGA) (Figure 19.5).v. Typical tRNA suppressor mutations are in redundant tRNA genes, so the wild-
type tRNA activity is not lost.
vi. Nonsense suppression occurs by competition between release factors andsuppressor tRNAs.
(1) UAG and UGA suppressor tRNAs do well in competition with release factors.
(2) UAA suppressor tRNAs are only 15% efficient.
vii. Suppression by a tRNA occurs at all of its specific stop codons (e.g., UGA orUAG), not just the mutant one. This may produce read-through proteins, but theyare not as common as expected, possibly due to tandem stop codons (e.g.,UAGUGA).
7/28/2019 Mutation 4th
17/40
tRNA suppressor gene mechanism for nonsense mutation
7/28/2019 Mutation 4th
18/40
New mutations are categorized as
inducedorspontaneous
Induced mutationsare defined as those thatarise after purposeful treatment withmutagens, environmental agents that areknown to increase the rate of mutations
Spontaneous mutationsare those that arisein the absence of known mutagen
treatment. They account for the"background rate" of mutation and arepresumably the ultimate source of naturalgenetic variation that is seen in populations.
7/28/2019 Mutation 4th
19/40
Spontaneous Mutations
1. All types of point mutations can occur spontaneously, duringS, G1 and G2 phases of the cell cycle, or by the movement of
transposons.
2. The spontaneous mutation rate in eukaryotes is between 10-
4-to-10-6 per gene per generation, and in bacteria and phages
10-5
-to-10-7
/gene/generation.a. Genetic constitution of the organism affects its mutation
rate.
i. In Drosophila, males and females of the same strain
have similar mutation rates.
ii. Flies of different strains, however, may have different
mutation rates.
b. Many spontaneous errors are corrected by the cellular
repair systems, and so do not become fixed in DNA.
7/28/2019 Mutation 4th
20/40
Different types DNA replication errors
Wobble-pairing
T-G, C-A, A-G, T-C
Normal pairing typically occurs in the next round of
replication; frequency of mutants in F2 is .GT pairs are targets for correction by proofreading andother repair systems
Additions and deletions
DNA loops out on template strand, DNA polymerase skipsbases, and deletion occurs
DNA loops out on new strand, DNA polymerase addsuntemplated bases
7/28/2019 Mutation 4th
21/40
Mutation caused by mismatch wobble base pairing
7/28/2019 Mutation 4th
22/40
Addition and deletion by DNA looping-out
7/28/2019 Mutation 4th
23/40
Spontaneous chemical changes
Depurination
Common; A or G are removed and replaced with arandom base.
Deamination
Amino group is removed from a base (C U); if notreplaced U pairs with A in next round of replication(CG TA).
Prokaryote DNA contains small amounts of 5MC;deamination of 5MC produces T (CG TA).
Regions with high levels of 5MC are mutation hotspots.
7/28/2019 Mutation 4th
24/40
Deamination
7/28/2019 Mutation 4th
25/40
Induced mutations
Radiation (e.g., X-rays, UV)
Ionizing radiation breaks covalent bonds includingthose in DNA and is the leading cause of chromosomemutations
Ionizing radiation has a cumulative effect and kills cellsat high doses
UV (254-260 nm) causes purines and pyrimidines to
form abnormal dimer bonds and bulges in the DNAstrands
7/28/2019 Mutation 4th
26/40
Thymine dimers induced by UV light.
7/28/2019 Mutation 4th
27/40
Induced mutations: chemical mutagens
Base analogs
Similar to normal bases, incorporated into DNA duringreplication
Some cause mis-pairing (e.g., 5-bromouracil)
Not all are mutagenic
7/28/2019 Mutation 4th
28/40
Mutagenic efffects
of 5-bromouracil
7/28/2019 Mutation 4th
29/40
Mutagenic efffects of 5-bromouracil
7/28/2019 Mutation 4th
30/40
Induced mutations: Chemical mutagens
Base modifying agents, act at any stage of
the cell cycle:
Deaminating agents
Hydroxylating agents
Alkylating agents
7/28/2019 Mutation 4th
31/40
Base-modifying agents
10.24
7/28/2019 Mutation 4th
32/40
Base-modifying agents (cont.).
10.25
7/28/2019 Mutation 4th
33/40
Induced mutations: chemical mutagens
Intercalating agents:
Thin, plate-like hydrophobicmolecules insert themselvesbetween adjacent base-pairs,
Mutagenic intercalatingagents cause insertionsduring DNA replication.
Loss of intercalating agentcan result in deletion.
Examples: proflavin, ethidiumbromide
7/28/2019 Mutation 4th
34/40
DNA Repair Mechanisms
1. Both prokaryotes and eukaryotes have
enzyme-based DNA repair systems that
prevent mutations and even death from
DNA damage.
2. Repair systems are grouped by their repair
mechanisms. Some directly correct, while
others excise the damaged area and then
repair the gap.
7/28/2019 Mutation 4th
35/40
Direct Correction of Mutational Lesions
1. DNA polymerase proofreading corrects most of the incorrect nucleotide
insertions that occur during DNA synthesis, which stalls until the wrongnucleotide is replaced with a correct one.
a. The role of 3-to-5 exonuclease activity is illustrated by mutator mutations in E.
coli, which confer a much higher mutation rate on the cells that carry them.
b. The mutD gene, encoding the e subunit of DNA polymerase III, is an example.
Cells mutant in mutD are defective in proofreading.
2. UV-induced pyrimidine dimers are repaired using photoreactivation (light
repair).
a. Near UV light (320370 nm) activates photolyase (product of the phr gene) to
split the dimer.
b. Photolyases are found in prokaryotes and simple eukaryotes, but not in
humans.
3. Damage by alkylation (usually methyl or ethyl groups) can be removed by
specific DNA repair enzymes.
a. For example, O6-methylguanine methyltransferase (from the ada gene)
recognizes O6-methylguanine in DNA, and removes the methyl group.
b. Demethylation restores the base to its original form.
7/28/2019 Mutation 4th
36/40
Repair Involving Excision of Base Pairs
1. Another repair system, which does not require light, was discovered
in 1964. It is called dark repair, the excision repair system, or thenucleotide excision repair (NER) system.
a. In E. coli, NER corrects pyrimidine dimers and other damage-induced distortions
of the DNA helix.
b. The proteins required are UvrA, UvrB, UvrC and UvrD (encoded by genes of the
same name) (Figure 19.17).
c. A complex of two UvrA and one UvrB proteins slides along the DNA. When it
encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the
UvrB at the lesion.
d. When UvrBC forms, the UvrC cuts 45 nucleotides from the lesion on the 3 side,
and eight nucleotides away on the 5 side. Then UvrB is released and UvrD binds
the 5 cut end.e. UvrD is a helicase that unwinds the region between the cuts, releasing the short
ssDNA, while DNA polymerase I fills the gap and DNA ligase seals the
backbone.
f. In yeast and mammalian systems, about 12 genes encode proteins involved in
excision repair.
7/28/2019 Mutation 4th
37/40
Nucleotide excision repair (NER) of pyrimidine dimmer
and other damage-induced distortions of DNA
7/28/2019 Mutation 4th
38/40
2. Methyl-directed mismatch repair recognizes mismatched base
pairs, excises the incorrect bases and then carries out repair
synthesis.
a. In E. coli, initial stages involve products of the mutS, mutL and mutHgenes.
i. MutS binds the mismatch, and determines which is the new strand by its
lack of methylation.
ii. MutL and MutH bind unmethylated GATC sequences (site of methylation
in E. coli) and bring the GATC close to the mismatch by binding MutS.
iii. MutH then nicks the unmethylated GATC site, the mismatch is removedby an exonuclease and the gap is repaired by DNA polymerase III and
ligase.
b. Eukaryotes also have mismatch repair, but it is not clear how old and new
DNA strands are identified.
i. Four genes are involved in humans, hMSH2 (homologous to E. coli
mutS), and hMLH1, hPMS1 and hPMS2(all homologous to mutL).ii. All of these are mutator genes, and mutation in any 1 of them confers
hereditary predisposition to hereditary nonpolyposis colon cancer(HNPCC).
7/28/2019 Mutation 4th
39/40
Mechanism of mismatch correction repair
7/28/2019 Mutation 4th
40/40
3. The SOS response in bacteria results when specific base pairing
cannot occur. It allows the cell to survive otherwise lethal events, but
usually at the cost of incurring new mutations.
a. E. coliSOS is controlled by two genes, lexA and recA. (Mutants in either
of these genes have their SOS response permanently turned on.)
i. When no DNA damage is present, LexA represses transcription of
about 17 genes with products involved in various types of DNA
repair.
ii. Sufficient DNA damage activates the RecA protein, which
stimulates LexA to autocleave, removing repression of the DNA
repair genes.
iii. After damage is repaired, RecA is inactivated, and newly
synthesized LexA again represses the DNA repair genes.
b. The SOS system is an error-prone bypass synthesis system.
i. Some lesions, like T^T dimers, are easily copied to give AA in the
new DNA strand.
ii. Others, like C^C dimers, stall the SOS repair system, creating a
delay during which C can be deaminated to a U (forming a C^U),
ti t iti t ti