Definitions:

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Definitions ? replication errors ? spontaneous
DNA damage ? DNA mutations ? double-strand break
(DSB) repair pathway
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Questions to be addressed

• How does the mismatch repair system accurately
  detect, remove and repair the mismatch resulting
  from inaccurate replication?
• What are the environmental factors that cause DNA
  damage?

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• How could a DNA damage be converted to DNA
  mutation?
• What are the mechanisms to repair a DNA damage?
  Describes how base excision repair and nucleotide
  excision repair work?
• What is translesion DNA synthesis? Why it is
  important?

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The mutability and repair of DNA

• Replication errors and their repair
• DNA damage
• Repair of DNA Damage

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Replication errors and their repair
Rapair
Mismatch repair
Proofreading
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The nature of mutations

• Simple mutations
• Transitions(pyrimidine-to-pyrimidine and
  purine-to-purine)
• Transversions(pyrimidine-purine and
  purine-to-pyrimidine)
• Insertions and deletions (a nucleotide or a small
  number of nucleotides)

?point mutations mutations that alter a single
nucleotide
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• Other kinds of mutation
• ----cause more drastic changes in DNA
• Extensive insertions and deletions
• Gross rearrangements
• Such changes might be caused by the insertion of
  a transposon or by the aberrant actions of
  cellular recombination processes

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hotspotssome sites on the chromosome where
mutations arise at high frequency while other
sites undergoing alterations at a comparatively
low frequency about 10-6 to 10-11 per round of
DNA. DNA microsatellites one kind of sequence
that is particularly prone to mutation merits
special comment , because of its importance in
human genetics and disease . They are repeats of
simple di-,tri- or tetranucleotide sequences,
which are known as DNA microsatellites.
(eg.dinucleotide sequence CA)
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The replication errors escape proofreading

• Proofreading improves the fidelity of DNA
  replication by a factor of about 100.The
  proofreading exonuclease is not poolproof.
• If the misincorporated nucleotide is not
  subsequently detected and replaced, the sequence
  change will become permanent in the genome.

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Mismatch repair removes errors that escape
proofreading

• ?Increase the accuracy of DNA by an
  additional 2-3 orders of magnitude
• ?Two challenges
• ?scan the genome rapidly
• ?correct the mismatch accurately ( that is it
  must recognize the newly synthesized strand)

E..coli
we take for example
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MutS scans the DNA, recognizing the mismatch from
the distortion they cause in the DNA backbone
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MutS embraces the mismatch-containing DNA,
inducing a pronounced kink in the DNA and a
conformational change in MutS itself. The complex
of MutS and the mismatch-containing DNA recruits
MurL. MutL,in turn, activates MutH, an enzyme
that causes an incision or nick on one strand
near the site of the mismatch. Necking is
followed by the action of a specific
helicase(UvrD) and one of three exonucleases.
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The helicase unwinds the DNA ,starting from the
incision and moving in the direction of the site
of the mismatch ,and the exonuclease
progressively digests the displaced
nucleotide. This action produces a single-strand
gap, which is then filled in by DNA polymerase?
and sealed with DNA ligase.
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ATP
The MutS protein of Escherichia coli MutS is
responsible for recognizing and binding to base
pair mismatches, and recruits other key proteins
(MutH and MutL) required for repair to the
mismatch site.

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?
How does the E..coli mismatch repair
system know which of the two mismatched
nucleotides to replace? Dam methylases tags the
parental strand by transient hemimethylation and
methylates A residues on both strands of the
sequence 5-GATC-3. MutH protein become
activated only when it is contacted by MutL and
MutS located at a nearby mismatch.
NEXT PAGE
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Different exonucleases are used to remove
single-stranded DNA between the nick created by
MutH and the mismatch. Its all depending on
whether MutH cuts the DNA on the 5 or the 3
side of the misincorporated nucleotide.
NEXT PAGE
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Unmethylated GATC is 5 of mutation
Unmethylated GATC is 3 of mutation
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When it comes to eukaryotic cells MSH MutS
homologs MLH or PMS MutL homologs Eukaryotes
have multiple MutS-like proteins with different
specificities.
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DNA damage

• Three reasons for DNA damage
• hydrolysis(??) and deamination(???)
• Alkylation, Oxidation, and Radiation
• base analogs and intercalating agents

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DNA undergoes damage spontaneously from
hydrolysis(??) and deamination(???)

• This is ironic since the proper structure of the
  double helix depends on an aqueous environment.

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Deamination C-U
Depurination ----gt an abasic site
Deamination of 5-mC----gtT
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DNA is damaged by Alkylation, Oxidation, and
Radiation
Often mispqir with thymine GC AT
Reactive oxygen species O2-, H2O2, OH
G modification (alkylation oxidation)
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Incapable of base-pairing and cause the DNA
polymerse to stop during replication
Thymine dimer by ultraviolet light
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Clastogenic ionizing radiation and agents like
bleomycin that cause DNA to break are said to be
clastogenic.
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Mutations are also caused by base analogs and
intercalating agents
Base analogues
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Intercalating agents which cause the deletion or
addition of a base pair or a few base pairs
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Repair of DNA damage

• The consequences of damage to DNA
• Impediments (not permanent)
• Mispairing(can cause permanent alteration)

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Direct reversal of DNA damage
Photoreactivation (the enzyme DNA potolyase
captures energy from light )
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Methyl group removal (a methyltransferase
removes the methyl group by transferring it to
one of its own cysteine residues)
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Base exicision repair enzymes remove damaged
bases by a base-flipping mechanism
Base excision repair Nucleotide excision repair
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Base excision pathway
A glycosylase acts by hydrolyzing the glycosidic
bond then DNA polymerase and DNA ligase restore
an intact strand
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Structure of a DNA-glycosylase complex DNA
glycosylase leision-specific and cells have
multiple DNA glycosylases with different
specificities
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?
How do DNA glycosylases detect
damaged bases while scanning the genome?
Due to the flexibility of DNA ,the damaged base
is flipped out so that it projects away from the
double helix

The DNA-glycosylase complexs diffuse laterally
along the minor groove of the DNA until a
specific kind of lesion is detected
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If a damaged base is not removed by base excision
before DNA replication
A fail-safe system
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Nucleotide excision repair enzymes cleave damaged
DNA on either side of the lesion
?recognize distortions ? a single-stranded gap
in the DNA ? DNA polymerase or ligase fill in
the gap.
Use the undamaged DNA as a template
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E..coli
When it comes to There are four proteins about
nucleotide exicision UvrA, UvrB, UvrC, UvrD
Look at the next picture for detail
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2
3
1
4
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1.UvrA and UvrB scan DNA to identify a distortion
2. UvrA leaves the complex,and UvrB melts DNA
locally round the distortion 3. UvrC forms a
complex with UvrB and creates nicks to the 5
side of the lesion 4. DNA helicase UvrD releases
the single stranded fragment from the duplex, and
DNA Pol I and ligase repair and seal the gap
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Transcription coupled DNA repair nucleotide
excision repair system is capable of rescuing RNA
polymerase that has been arrested by the presence
of lesions in the DNA template
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Recombination repairs DNA breaks by retrieving
sequence information from undamaged DNA

• This is accomplished by the double-strand break
  (DSB) repair pathway

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Damage in the DNA template can lead to DSB
formation during replication
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• DSB repair model for homologous
• recombination

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Translesion DNA synthesis enables replication to
proceed across DNA damage

• Occurs when the above repairs are not efficient
  enough
• a fail-safe or last resort mechanism, which
  spares the cell the worse fate of an incompletely
  replicated chromosome

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Translesion DNA synthesis Catlyzed by a
specialized class of DMA polymerases that
synthesize DNA directily across the site of the
damage
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Crustal structure of a translesion polymerase
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The enzyme is not reading sequence
information from the template
SOS response
Translesion synthesis is often highly
error-prone
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THAT'S ALL