Chapter 9 The Mutability and Repair of DNA

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Chapter 9 The Mutability and Repair of DNA
Outline

• 1.replication errors and their repair
• 2. DNA damage
• 3.Repair of DNA damage

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Section 1replication errors and their repair

• Important definations
• Transitionsa kind of the simplest mutations
  which are pyrimidine-to-pyrimidine and
  purine-to-purine substitutions
• Tranversionsthe other kind of mutation which are
  pyrimidine-to-purine and purine-to-pyrimidine
  substitutions

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• Point mutationsmutations that alter a single
  nucleotide
• DNA microsatellites Mutation-prone sequence in
  human genome are repeats of simple di-, tri- or
  tetranucleotide sequences, known as DNA
  microsatellites

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The Nature of Mutations
Base change substitutions
transitions
transversions
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• Other kinds of mutations(which cause more
  drastic changes in DNA)
• Insertions
• Deletions
• Gross rearrangements of chromosome

These mutations might be caused by insertion by
transposon or by aberrant action of cellular
recombination processes.
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Some Replication Errors Escape Proofreading

• The 3-5 exonuclease component of replisome
  only improves the fidelity of DNA replication by
  a factor of about 100.
• But, thats not enough
• The misincorporated nucleotide needs to be
  detected and replaced, otherwise it will cause
  mutation.

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A mutation may be introduced by misincorporation
of a base in the first round of replication.In
the second round of replication,the mutation
becomes permanently incorporated in the DNA
sequence.
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Mismatch Repair Removes Errors That Escape
Proofreading

• Mismatch repair systema system that increases
  the accuracy of DNA synthesis by an additional
  two to three orders of magnitude.
• This system faces 2 challenges(1)rapidly find
  the mismatches/mispairs
• (2) Accurately correct the
  mismatch

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Important parts of mismatch repair system

• MutSa dimer of the mismatch repair protein which
  detects mismatches

Fuctions of MutS 1. MutS scans the DNA,
recognizes the mismatch from the distortion they
cause in the DNA backbone
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Functions of MutS

• 2.MutS embraces the mismatch-containing DNA,
  inducing a pronounced kink in the DNA and a
  conformational change in MutS itself

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Crystal structure of MutS
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Further steps of miamatch repair,we must pay
attention to the other two important parts of the
mismatch repair system---MutL and MutH
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How these three parts interact

• MutS-mismatch-containing DNA complex recruits
  MutL, MutL in turn activates MutH, an enzyme
  causing an incision or nick on one strand near
  the site of the mismatch. Nicking is followed by
  the specific helicase and one of three
  exonucleases.

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Then we talk about

• how does the E.coli mismatch repair system
  know which of the two mismatched nucleotides to
  replace?

(Dam)methylation
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• Dam methylasethe E.coli enzyme that methylases A
  residues on both strands of the sequence
  5-GATC-3.
• The newly synthesized strand is not methylated
  by Dam methylase in a few minutes after the
  synthesis.

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a.Replication generates hemimethylated DNA in
E.coli. b.MutH makes incision in unmethylated
daughter strand.
Dam methylation at replication fork
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Different exonucleases are used to remove
single-strand DNA between the nick created by
MutH and the mismatch.
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Eukaryotic cells

• In fact,eukaryotes have multiple MutS-like
  proteins with different specificities.
• MSH proteins MutS homologs

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Section 2DNA Damage

• There are mainly three kinds of ways that DNA is
  damaged
• DNA undergoes damage spontaneously from
  hydrolysis and deamination
• DNA is damaged by Alkylation,Oxidation and
  Radiation
• Mutations are also caused by base analogs and
  intercalating agents

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1st kind, Hydrolysis Deamination
a.Deamination that Cytosine to Uracil which
explain why DNA contains T instead of U
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• Answer
• If DNA naturally contained uracil instead of
  thymine,the deamination of cytosine will create a
  natrual base which the repair system will not
  easily recognize.

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2nd kind,Alkylation Oxidation and Radiation
DNA is subject to attack from Reactive oxygen
species (O2-, H2O2, OH)
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UV(???) induces a cyclobutane between adjacent
thymines
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3rd kind,base analogs intercalating agents

• Base analogs similar enough to the normal bases
  to be processed by cells and incorporated into
  DNA during replication.
• But they base pair differently, leading to
  mistake during replication.
• The most mutagenic base anolog is
  5-bromouracil,an anolog of thymine.

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5-bromouracil,base anolog of thymine,can mispair
with guanine
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Intercalating agents

• Intercalating agents are flat molecules
  containing several polycyclic rings that interact
  with the normal bases in DNA through hydrogen
  bonds and base stacking.

???
???
???
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Section 3Repair of DNA Damage

• There are two consequences of DNA damage
• Some kinds of damage create impediments to
  replication or transcription
• Other kinds of damage create altered bases that
  cause mispairing which results a permanent
  alternation to DNA

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Systems that repair damage to DNA

• A repair enzyme simply reverses the damage
• Excision repair systems,in which damaged
  nucleotide is not repaired but removed from
  DNA(more elaborate step),composed of base
  excision repair and nucleotide excision repair

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Systems that repair damage to DNA

• Recombinational repair,which is employed when
  both strands of DNA are damaged,also known as
  double-strand break repair.(more elaborate)
• Translesion DNA synthesis,the last way cells
  choose

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Direct reversal of DNA damage
For example,photoreactivation,which directly
reverses pyrimidine dimers
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Direct reversal od DNA damage
Another example,methyltransferase(?????) directly
removes the methyl group from the O6-guanine
residue
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Base excision repair systems

• Base excision repair enzymesglycosylase(????)
  recognize and remove damaged bases by a
  base-flipping mechanism,hydrolyzing the
  glycosidic bond.
• DNA glycosylases are lesion-specific.

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1.The AP site is created by the hydrolysis of
glycosylase bond. 2.AP endonucleaseexonuclease
cut out the 5 phosphate. 3.DNA polymerase fill
in the gap.
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The enzyme
The damaged base which is filpped out
The DNA
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Nucleotide excision repair systems

• What is the difference between the two kinds of
  excision repair systems?
• Also,how does the nucleotidework?
• Recognize distortions to the shape of the DNA
  double helix
• Remove a short single-stranded segment that
  includes the lesion.
• DNA polymerase/ligase fill in the gap.

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Once encountering a distortion UvrA exits the
complex and UvrB melts the DNA to create a
single-strand bubble around the lesion.
Next,UvrB recruits UvrC,and UvrC creates two
incisions in different positions on one strand.
Finally,DNA polymerase and ligase fill in the
gap.
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Recombinational repair

• This is the very essencial way that cells repair
  double-strand breaks in DNA in which both strands
  of the duplex are broken.
• We call it double-strand break(DSB)repair
  pathway,which retrieve sequence information from
  sister chromosome.

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Translesion DNA synthesis

• When cells cannot repair certain
  lesions,there is a fail-safe mechanism that
  allows the replication machinery to bypass these
  sites of damage----translesion synthesis
• Translesion synthesis is catalyzed by a
  specialized class of DNA polymerases that
  synthesize DNA directly across the damage site.
• Translesion polymerase is produced by cell in
  response to the DNA damage
• Translesion polymerases are expressed as part of
  the SOS response pathway.

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Crystal structure of a translesion polymerase.
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Translesion DNA synthesis in E. coli
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