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Chapter 12
Molecular Mechanisms of
Mutation and DNA Repair
Mutations
• A mutation is any heritable change in the genetic
material
• Mutations are classified in a variety of ways
• Most mutations are spontaneous—they are
random, unpredictable events
• Each gene has a characteristic rate of spontaneous
mutation, measured as the probability of a change
in DNA sequence in the time span of a single
generation
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Table 12.1
Table 12.1: Major types of mutations and their distinguishing features
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Mutations
• Rates of mutation can be increased by treatment
with a chemical mutagen or radiation, in which case
the mutations are said to be induced
• Mutations in cells that form gametes are germ-line
mutations; all others are somatic mutations
• Germ-line mutations are inherited; somatic
mutations are not
• A somatic mutation yields an organism that is
genotypically a mixture (mosaic) of normal and
mutant tissue
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Mutations
• Among the mutations that are most useful for
genetic analysis are those whose effects can be
turned on or off by the researcher
• These are conditional mutations: they produce
phenotypic changes under specific (permissive
conditions) conditions but not others (restrictive
conditions)
• Temperature-sensitive mutations: conditional
mutation whose expression depends on
temperature
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Figure 12.1: Siamese cat
Courtesy of Jen Vertullo
Mutations
• Mutations can also be classified according to their
effects on gene function:
 A loss-of-function mutation (a knockout or null) results in
complete gene inactivation or in a completely nonfunctional
gene product
 A hypomorphic mutation reduces the level of expression of a
gene or activity of a product
 A hypermorphic mutation produces a greater-than-normal
level of gene expression because it changes the regulation of
the gene so that the gene product is overproduced
 A gain-of-function mutation qualitatively alters the action of a
gene. For example, a gain-of-function mutation may cause a
gene to become active in a type of cell or tissue in which the
gene is not normally active.
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Figure 02A: An adult head in which both antennae form eye structures
Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995):
1788-1792. Reprinted with permission from AAAS.
[http://www.sciencemag.org/].
Figure 02B: A wing with eye tissue growing out from it
Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995):
1788-1792. Reprinted with permission from AAAS.
[http://www.sciencemag.org/].
Figure 02C: A single antenna in which most of the third segment consists of eye
tissue
Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995):
1788-1792. Reprinted with permission from AAAS.
[http://www.sciencemag.org/].
Figure 02D: Middle leg with an eye outgrowth at the base of the tibia
Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995):
1788-1792. Reprinted with permission from AAAS.
[http://www.sciencemag.org/].
Mutations
• Mutations result from changes in DNA
•
A base substitution replaces one nucleotide pair
with another
• Transition mutations replace one pyrimidine base
with the other or one purine base with the other.
There are four possible transition mutations
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Mutations
• Transversion mutations replace a pyrimidine
with a purine or the other way around. There are
eight possible transversion mutations
• Spontaneous base substitutions are biased in
favor of transitions
• Among spontaneous base substitutions, the
ratio of transitions to transversions is
approximately 2:1
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Mutations
• Mutations in protein-coding regions can change an
amino acid, truncate the protein, or shift the
reading frame:
• Missense or nonsynonymous substitutions result
in one amino acid being replaced with another
• Synonymous or silent substitutions in DNA do not
change the amino acid sequence
• Silent mutations are possible because the genetic
code is redundant
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Mutations
• A nonsense mutation creates a new stop codon
• Frameshift mutations shift the reading frame of
the codons in the mRNA
• Any addition or deletion that is not a multiple of
three nucleotides will produce a frameshift
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Sickle-cell anemia
• The molecular basis of sickle-cell anemia is a mutant
gene for b-globin
• The sickle-cell mutation changes the sixth codon in
the coding sequence from the normal GAG, which
codes for glutamic acid, into the codon GUG, which
codes for valine
• Sickle-cell anemia is a severe genetic disease that
often results in premature death
• The disease is very common in regions where malaria
is widespread because it confers resistance to malaria
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Figure 12.3: Molecular basis of sickle-cell anemia
Trinucleotide repeats
• Genetic studies of an X-linked form of mental
retardation revealed a class of mutations called
dynamic mutations because of the extraordinary
genetic instability of the region of DNA involved
• The molecular basis of genetic instability is a
trinucleotide repeat expansion due to the process
called replication slippage
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Figure 12.6: Model of replication slippage
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Fragile-X Syndrome
• The X-linked condition, is associated with a class of X
chromosomes that tends to fracture in cultured cells
that are starved for DNA precursors
• They are called fragile-X chromosomes, and the
associated form of mental retardation is the fragile-X
syndrome
• The fragile-X syndrome affects about 1 in 2500 children
• The molecular basis of the fragile-X chromosome has
been traced to the expansion of a CGG trinucleotide
repeat present at the site where the breakage takes
place
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Figure 12.4: Pedigree showing transmission of the fragile-X syndrome
Adapted from C. D. Laird, Genetics 117 (1987): 587-599.
Fragile-X Syndrome
• Normal X chromosomes have 6–54 tandem copies of
CGG, whereas affected persons have 230–2300 or
more copies
• An excessive number of copies of the CGG repeat
cause loss of function of a gene designated
FMR1(fragile-site mental retardation-1)
• Most fragile-X patients exhibit no FMR1 mRNA
• The FMR1 gene is expressed primarily in the brain
and testes
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Figure 12.5: Dynamic mutation
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Dynamic Mutations and Diseases
• Other genetic diseases associated with dynamic
mutation include:
 The neurological disorders myotonic
dystrophy (with an unstable repeat of CTG)
 Kennedy disease (AGC)
 Friedreich ataxia (AAG)
 Spinocerebellar ataxia type 1 (AGC)
 Huntington disease (AGC)
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Transposable Elements
• In a 1940s study of the genetics of kernel mottling in
maize, Barbara McClintock discovered a genetic
element that could move (transpose) within the
genome and also caused modification in the
expression of genes at or near its insertion site.
• Since then, many transposable elements (TEs) have
been discovered in prokaryotes and eukaryotes
• They are grouped into “families” based on similarity
in DNA sequence
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Transposable Elements
• The genomes of most organisms contain multiple
copies of each of several distinct families of TEs
• Once situated in the genome, TEs can persist for
long periods and undergo multiple mutational
changes
• Approximately 50% of the human genome consists
of TEs; most of them are evolutionary remnants no
longer able to transpose
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Transposable Elements
• Some transposable elements transpose via a DNA
intermediate others via an RNA intermediate
• A target-site duplication is characteristic of most
TEs insertions, and it results from asymmetrical
cleavage of the target sequence
• A large class of TEs called DNA transposons
transpose via a cut-and-paste mechanism: the TE is
cleaved from one position in the genome and the
same molecule is inserted somewhere else
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Figure 12.8: The sequence arrangement of a cut-and-paste transposable
element and the changes that take place when it inserts into the genome 28
Transposable Elements
• Each family of TEs has its own transposase—an
enzyme that determines distance between the
cuts made in the target DNA strands
• Characteristic of DNA TEs is the presence of
short terminal inverted repeats
• Another large class of TEs possess terminal
direct repeats, 200–500 bp in length, called long
terminal repeats, or LTRs
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Transposable Elements
• TEs with long terminal repeats are called LTR
retrotransposons because they transpose using an
RNA transcript as an intermediate
• Among the encoded proteins is an enzyme known
as reverse transcriptase, which can “reversetranscribe,” using the RNA transcript as a template
for making a complementary DNA daughter strand
• Some retrotransposable elements have no terminal
repeats and are called non-LTR retrotransposons
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Figure 12.10: Drosophila melanogaster
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Transposable Elements
• TEs can cause mutations by insertion or by
recombination
• In Drosophila, about half of all spontaneous
mutations that have visible phenotypic effects
result from insertions of TEs
• Genetic aberrations can also be caused by
recombination between different (nonallelic)
copies of a TE
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Figure 12.11: Recombination between transposable elements
Figure 12.12: Unequal crossing-over
Spontaneous Mutations
• Mutations are statistically random events—there
is no way of predicting when, or in which cell, a
mutation will take place
• The mutational process is also random in the
sense that whether a particular mutation
happens is unrelated to any adaptive advantage
it may confer on the organism in its environment
• A potentially favorable mutation does not arise
because the organism has a need for it
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Spontaneous Mutations
• Several types of experiments showed that
adaptive mutations take place spontaneously and
were present at low frequency in the population
even before it was exposed to the selective agent
• One experiment utilized a technique developed by
Joshua and Esther Lederberg called replica plating
• Selective techniques merely select mutants that
preexist in a population
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Figure 12.13: Replica plating
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Figure1 2.14: The ClB method for estimating the rate at which spontaneous
recessive lethal mutations arise
Mutation Hot Spots
• Mutations are nonrandom with respect to position
in a gene or genome
• Certain DNA sequences are called mutational
hotspots because they are more likely to undergo
mutation than others
• For instance, sites of cytosine methylation are
usually highly mutable
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Figure 12.15: Spontaneous loss of the amino group
Mutagenes
• Almost any kind of mutation that can be induced by a mutagen can
also occur spontaneously, but mutagens bias the types of
mutations that occur according to the type of damage to the DNA
that they produce
Table 12.3: Major agents of mutation and their mechanisms of action
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Figure 12.16: Depurination
Figure 12.17: Deamination of adenine results in hypoxanthine
Figure 12.18: Mispairing mutagenesis by 5-bromouracil
Figure 12.19: Two pathways for mutagenesis by 5-bromouracil (Bu)
Figure 12.20: Chemical structures of two highly mutagenic alkylating agents
Figure 12.21: Mutagenesis of guanine by ethyl methanesulfonate (EMS)
Figure 12.22: Structural view of the formation of a thymine dimer
Figure 12.25: Mutation rates of five tandem repeats
Data from Y. E. Dubrova, et al., Nature 380 (1996): 683-686.
DNA Repair Mechanisms
• Many types of DNA damage can be repaired
• Mismatch repair fixes incorrectly matched base
pairs
• The AP endonuclease system repairs nucleotide
sites at which the base has been lost
• Special enzymes repair damage caused to DNA by
ultraviolet light
• Excision repair works on a wide variety of
damaged DNA
• Postreplication repair skips over damaged bases
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Mismatch Repair
• Mismatch repair fixes incorrectly matched base
pairs: a segment of DNA that contains a base
mismatch excised and repair synthesis followed
• The mismatch-repair system recognizes the
degree of methylation of a strand and
preferentially excises nucleotides from the
undermethylated strand
• This helps ensure that incorrect nucleotides
incorporated into the daughter strand in
replication will be removed and repaired.
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Table 12.6: Types of DNA damage and mechanism of repair
Mismatch Repair
• The most important role
of mismatch repair is as
a “last chance” errorcorrecting mechanism in
replication
Figure 12.26: Summary of rates of
error in DNA polymerization,
proofreading, and postreplication
mismatch repair
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Mismatch Repair
• The daughter strand is
always the undermethylated
strand because its
methylation lags somewhat
behind the moving
replication fork
Figure 12.27: Mismatch repair
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AP Repair
• Deamination of cytosine creates uracil, which is
removed by DNA uracil glycosylase from
deoxyribose sugar. The result is a site in the DNA
that lacks a pyrimidine base (an apyrimidinic site)
• Purines in DNA are somewhat prone to hydrolysis,
which leave a site that is lacking a purine base (an
apurinic site)
• Both apyrimidinic and apurinic sites are repaired
by a system that depends on an enzyme called AP
endonuclease
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Figure 12.28: Base-excision
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Figure 12.29: Action of AP endonuclease
Excision Repair
• Excision repair is a
ubiquitous, multistep
enzymatic process by
which a stretch of a
damaged DNA strand
is removed from a
duplex molecule and
replaced by
resynthesis using the
undamaged strand as
a template
Figure 12.30: Mechanism of nucleotide excision repair of damage to DNA
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Postreplication repair
• Sometimes DNA
damage persists rather
than being reversed or
removed, but its
harmful effects may be
minimized. This often
requires replication
across damaged areas,
so the process is called
postreplication repair
Figure 12.31: Postreplication repair
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Ames Test
• In view of the increased number of chemicals used
and present as environmental contaminants, tests
for the mutagenicity of these substances has
become important
• Furthermore, most agents that cause cancer
(carcinogens) are also mutagens, and so
mutagenicity provides an initial screening for
potential hazardous agents
• A genetic test for mutations in bacteria that is
widely used for the detection of chemical mutagens
is the Ames test
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Ames test
• In the Ames test for mutation, histidine-requiring
(His-) mutants of the bacterium Salmonella
typhimurium, containing either a base substitution
or a frameshift mutation, are tested for
backmutation reversion to His+
• In addition, the bacterial strains have been made
more sensitive to mutagenesis by the incorporation
of several mutant alleles that inactivate the excisionrepair system and that make the cells more
permeable to foreign molecules
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