DNA Mutation And Repair Base Mismatch Repair DNA polymerase

1
DNA Mutation And Repair
2
A Genes Mutation Rate Is Influenced By Several
Factors
Spontaneous DNA mutation rate (varies from gene
to gene)
Exposure to dietary, environmental and lifestyle
factors
Capacity to repair mutations
Some mutations convey advantages in some
environments
Stressful environment may increase mutation rate
3
DNA Mutation Terminology
Loss-of-function mutations impair the function of
the protein
Gain-of-function mutations cause the protein to
be expressed in tissues where it isnt supposed
to be expressed
Germline mutationsPresent in either (or both)
the sperm and egg that made the individual,
therefore present in every cell the individual has
Somatic mutationsArise after fertilization,
during cell replication/division/differentiation/m
igration, therefore only present in a subset of
the individuals cells
4
DNA Mutation Terminology
Conditional mutationSome mutations only cause
consequences under certain conditions, such as
excessively high temperatures Ex. Some mutations
in genes whose proteins metabolize food will only
exert an effect on the individual after he/she
eats certain foods
Forward mutationchanges the phenotype (most
mutations)
Reverse mutation, aka reversionchanges the
mutant genotype back into the wild-type genotype
Splice mutationA mutation that affects the
pattern of RNA splicing, thereby changing the
content of the mRNA
Frameshift mutationA mutation that shifts the
ribosomes reading frame, by inserting or
deleting nucleotides in the mRNA
5
Substitutions, Insertions And Deletions Are Most
Common
Unless it is an in-frame del/ins, i.e. multiple
of 3
6
Single-Nucleotide Substitutions Often Arise
Because Of DNA Polymerase Errors
Transition purine-purine substitution or
pyrimidine-pyrimidine substitution Transversion
purine-pyrimidine substitution or vice versa
Missense mutationcauses one amino acid to
replace another
Nonsense mutationcreates a STOP codon at the
site of the mutation
Neutral mutationchanges the amino acid content
of the protein, but has no functional consequences
Silent, aka synonymous, mutationdoes not change
the amino acid content of the protein
7
Single-Nucleotide Substitutions Often Arise
Because Of DNA Polymerase Errors
Functional consequences vary No change in
protein activity benign polymorphisms, aka
single nucleotide polymorphisms, aka SNPs Those
that alter protein activity may increase or
decrease it, by a small or large amount
Note that silent mutations may not be
silentthey may change the splicing of the mRNA
(intronic mutations may also) Recall that almost
all human introns begin with GT If a C ?T
mutation changes a GC near the end of an exon to
a GT, the spliceosome may splice after that GT
8
Insertions And Deletions May Arise Through
Strand Slippage
9
Repeated Sequences May Cause Insertions And
Deletions Through Unequal Crossovers
During metaphase I of meiosis
10
Trinucleotide Repeats Expand
Expansions are most common, but sometimes the
repeat can contract Whether it expands or
contracts may depend on the parent of origin
The larger it gets, the more it expands from one
generation to the next One indiv 60
repeats child 250 repeats grandchild 5,000
repeats
11
Fragile X Syndrome Is Due To An Expanding CCG
Repeat In The X Chromosome (Xq28)
12
Fragile X Syndrome
13
Trinucleotide Repeat Expansion Causes
Anticipation At The Clinical Level
14
Suppressor Mutations
Suppressor mutations can hide or suppress the
effects of other mutations The individual is a
double mutant, but has a normal phenotype Some
suppressor mutations are intragenicwithin the
same genes coding sequence
15
Suppressor Mutations
Some suppressor mutations are intergenicin a
second genes coding sequence
Ex. Nonsense mutation is suppressed by mutation
in a tRNA gene
16
Types Of DNA Mutations
Ex. Nonsense mutation is suppressed by mutation
in a tRNA gene
If multiple tRNAs carry this amino acid, the
other tRNAs will compensate for the fact that
this tRNA is mutant
This will cause a partial failure to stop
transcription in other genes with the same STOP
codon
17
Forward, Reverse And Suppressor Mutations
Reverse mutation, aka reversion, restores the
wild-type genotype Suppressor compensates for
the mutation
18
DNA Damage And Repair
Spontaneous Mutations Errors during DNA
replication Hydrolytic and other reactions
can cause base changes in DNA after
replication
Induced Mutations Environmental agents such
as UV light or radon Chemical exposures
Metabolic byproducts such as the superoxide ion
O2-
19
Incorporated Errors Errors Arising During DNA
Replication
Most common mechanism promiscuous basepairing
enabled by the flexibility in the DNA structure
20
Incorporated Errors Lead To Replication Errors
During the next cell cycle, the strands with the
mismatched bases serve as templates for DNA
replication
Replication proceeds properly, resulting in one
of the two new chromatids having the wrong base
on both strands of its DNA
21
Spontaneous Chemical Modification Of DNAEx.
Depurination
Hydrolysis reactions remove purine (A and G)
rings by cleaving the N-glycosidic bond that
holds them to the sugar
An adenine-containing nucleotide is usually
incorporated across from the depurinated one
during the next round of replication
22
Spontaneous Chemical Modification Of DNAEx.
Deamination
Pyrimidines (C, U and T) can be deaminated by
hydrolysis of the NH2 group the reaction doesnt
even require an enzyme Some Cs exist in
methylated form they get deaminated to yield
Ts Unmethylated Cs simply get deaminated to
yield Us.
23
Modification Of DNA By Chemical Agents (Induced
Mutations)
Deamination can also result from chemical
agents Nitrous acid HNO2 deaminates C to yield
U, which pairs with A, instead of the G
that C should pair with.
24
Induced Mutations Resulting From Chemical Agents
Alkylating agents can add methyl groups, ex.
Change guanine to methylguanine, which pairs
with T, instead of C.
25
Induced Mutations Resulting From Chemical Agents
Heterocyclic base analogs, ex. 5-bromouracil, can
cause base mispairings
The addition of the Br causes the U to want to
form three hydrogen bonds, instead of the usual
two
26
Induced Mutations Resulting From Chemical Agents
Reactive oxygen species (superoxide ion O2- or
hydrogen peroxide H2O2) are created by
normal metabolism. These chemicals oxidize
guanine to 8-oxoguanine, which pairs with A,
rather than C, as G should
27
Induced Mutations Resulting From Chemical Agents
Intercalating agents insert themselves between
nucleotides, increasing the distance between
stacked bases, confusing the DNA polymerase and
causing insertions and deletions in the DNA
28
Ionizing Radiation Breaks The DNA
Ionizing radiation (from X rays or radon in
homes) causes many molecules to form ions and
highly reactive free radicals These free
radicals can cause breaks in the DNA
29
The Suns UV Rays Cause Pyrimidine Dimers
Bonds form between neighboring Cs or Ts in the
same DNA strand This brings the two neighboring
nucleotides closer together, leaving the bases
unable to bond with the bases from the other DNA
strand
A C T T G C T G A A C G
A C T T G C T G A A C G
UV rays
This prevents DNA replication, which prevents the
cell from going through the cell cycle, causing
the cell to go into apoptosis This is why UV
light kills bacteria and is a good water
sterilizer
Bacteria can sometimes use the SOS system to
overcome the block in replication and
survive Eukaryotes have a special DNA polymerase
eta that puts AA across from the pyrimidine dimer
30
The Ames Test For Mutagenicity
The Ames test uses four strains of his-
Salmonella Typhimurium bacteria, which cannot
synthesize histidine and are vulnerable to
chemical mutagens, because they have loss-of
function mutations in genes whose
proteins Synthesize the amino acid
histidine Synthesize the lipopolysaccharide coat
that protects the bacteria from environmental
chemicals Repair DNA mismatches and damage
The bacteria are provided a limited amount of
histidine at first, but in order to survive, the
bacteria must acquire a reverse mutation in one
of the histidine-synthesizing genes that restores
the wild-type his phenotype
31
The Ames Test For Mutagenicity
The four strains of his- Salmonella bacteria have
different mutations in the histidine-synthesizing
genes One has a single nucleotide
substitution Three have frameshift mutations
Some mutagens produce reversions in just one
strain, giving a clue as to their mechanism of
action
32
Mechanisms For DNA Repair
33
DNA Repair Mechanisms Are Your Personal Unsung
Heroes
Two types of abnormalities need repair Base
mismatches Damage to the structure of the DNA
itself, ex. breaks in the chromosome or
pyrimidine dimers
There are several different types of repair
system, and most mismatch/damage can be corrected
by more than one system It is estimated that
approximately 99.9 of the mismatches and damage
that get done to your DNA gets repaired
It is common to find a great many gene mutations
and chromosome abnormalities in cancer
cells Losing the ability to repair DNA
mismatches and damage is thought to be one of the
critical steps in allowing a cell to become
malignant
34
(No Transcript)
35
Base Mismatch Repair
DNA polymerase proofreads itself, but makes an
error every approx. 10,000-100,000 nucleotides
(104 105) After repair, the frequency of
errant nucleotides is only approx. 1 in 1 billion
(109) nucleotides
Mismatched bases and loops such as those that
lead to deletions and duplications form bubbles
in the DNA double helix, which are recognized by
the repair systems
36
Mismatch Repair In Escherischia coli
37
Direct Repair
Alkylation can be reversed by O6-methylguanine
DNA methyltransferase
In bacteria, photolyase breaks the bonds that
maintain pyrimidine dimers
38
Deaminations Are Repaired By Base Excision
39
Deaminations Are Repaired By Base Excision
40
Deaminations Are Repaired By Base Excision
41
Deaminations Are Repaired By Base Excision
42
Base Excision Repair Is Error-Prone
In eukaryotes, DNA polymerase beta, which has no
proofreading ability, fills in the gap DNA
polymerase betas error rate is high enough to
leave approx. 10 new mutations uncorrected per
day The AP endonuclease already mentioned
proofreads
43
Nucleotide Excision Repairs Pyrimidine Dimers And
Large Distortions Of The DNA Helix
endonuclease
44
Nucleotide Excision Repair Is Deficient In
Xeroderma Pigmentosum
45
Genetic Disorders Resulting From Deficiencies In
DNA Repair