Transcript Ch. 13: Presentation Slides

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
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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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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
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normally active.
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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Fig. 12.19
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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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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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Fig. 12.6
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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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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 brain and
testes
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Fig. 12.5
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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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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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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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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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Fig. 12.13
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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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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
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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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Mismatch
Repair
• The daughter strand
is always the
undermethylated
strand because its
methylation lags
somewhat behind
the moving
replication fork
Fig. 12.27
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Mismatch Repair
• The most important
role of mismatch
repair is as a “last
chance” errorcorrecting
mechanism in
replication
Fig. 12.26
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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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Fig. 12.28
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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
Fig. 12.29
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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
Fig. 12.30
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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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