Mismatch Repair

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Mismatch Repair

• Sreeparna Banerjee, PhD
• Room Z 16 Ext 6468
• banerjee_at_metu.edu.tr

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• Topics covered
• Prokaryotic methyl directed mismatch repair (MMR)
• Eukaryotic MMR
• Role of MMR in homologous recombination
• Role of MMR in meiosis
• MMR and oxidative DNA damage/alkylation damage
  and drug tolerance
• MMR and the human factor (Lynch syndrome)

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• Prokaryotic MMR

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Paul Modrich the pioneer of methyl directed MMR
in E. coli
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What is repaired?

• Base-base mismatches (misincorporation)
• G.T, A.C gt G.G A.A gt T.T, C.T., G.A gt C.C (not
  repaired)
• Insertion deletion loops (polymerase slippage)

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Bidirectional MMR in E. coli
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The mechanism

• The mispair is recognized by MutS. MutL is
  recruited.
• MutH endonuclease activated in presence of MutS,
  MutL, a mismatch and ATP hydrolysis
• The activated MutH cleaves the unmodified strand
  at a GATC site that can be on either side of the
  mismatch.
• MutS and MutL help to load DNA helicase II (uvrD
  gene product) in a biased fashion so that it
  unwinds towards the mismatch.
• Excision takes place in the region spanning the
  two sites.
• Repair synthesis is initiated near the GATC site
  or the mismatch, depending on the polarity of the
  unmodified strand.

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MutS

• MutS (853aa, 95kD) forms dimers shaped like a
  fenestrated disk that encircles DNA containing a
  mispair or insertion deletion loop.
• The N-terminal domain is involved in DNA binding
• C-term involved dimerization and binding to MutL.
• C-term also possesses an asymmetric ATP binding
  and hydrolysis activity.
• Conserved Phe residue at the DNA binding site
  stacks against the mispaired base (T of a G.T
  mispair) or IDL.
• May also form H bonds with unpaired bases.
• The affinity of binding correlates with the
  efficiency with which different proteins are
  repaired (G.T highest, C.C not repaired)

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Crystal structure of Taq MutS
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MutH

• 25kD monomeric protein with a weak endonuclease
  activity in isolation.
• In presence of MutS, MutL, mispair ternary
  complex the endonuclease activity is greatly
  stimulated.
• Nicks one strand of at unmethylated GATC sequence
  5 or 3 to the mispair.

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MutL

• Forms a ring shaped dimer.
• Probably interacts with one subunit of MutS.
• Bridges between MutS and MutH.
• Has an N-term ATPase domain which can bind non
  specifically to DNA and stimulate MutH to cleave
  at the GATC site
• ATP binding and not hydrolysis is required for
  stimulation of MutH endonuclease.

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Helicase II (UvrD)

• Stimulated by MutL in a mismatch dependent
  manner.
• Translocates along DNA in a 3 5 direction in
  an ATP driven manner and unwinds towards the
  mismatch.
• Loaded at the GATC incision when the nick is 3
  to the mismatch and on the continuous strand when
  the nick is 5 to the incision.
• Subsequent degradation of the unwound strand is
  carried out by the 4 exonucleases
• ExoVII and RecJ- 5 3 exonuclease and
• ExoI and ExoX 3 5 exonuclease

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MMR assay
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(A) MutS binds to DNA nonspecifically and bends
it in search of a mismatch (B) upon specific
recognition of a mismatch, MutS undergoes a
conformational change to an initial recognition
complex (IRC) in which the DNA is kinked, with
interactions similar to those in the crystal
structures and (C) MutS then undergoes further
conformational changes to the ultimate
recognition complex (URC) in which the DNA is
unbent with the mismatched base possibly being
flipped out.
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Eukaryotic Mismatch Repair
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Richard Kolodner Harvard Medical School
Boston Ludwig Cancer Research Center UCSD San
Diego
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Eukaryotic MMR
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MutS homologs

• 6 homologs in S. Cerevisiae (MSH1-MSH6)
• MSH1 protein targeted to yeast mitochondria (no
  homolog in humans). Acts as a backup to
  mitochondrial BER for repair of oxidative damage
  to mitDNA
• MSH2, MSH6 involved in MMR
• MSH3, MSH6 involved in MMR
• MSH4 AND MSH5 involved in meiosis (crossing over
• during meiotic recombination)
• MSH6 was initially called GTBP or p160 in humans

Nuclear localization
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MutL homologs

• Four MutL homologs have been identified in
  eukaryotes (MSH1-3 and PMS1)
• MLH1-PMS1 (PMS2 in humans) involved in MMR
  interacts with MSH2.
• MLH1-MLH3 repair insertion deletion loops via the
  MSH2-MSH3 pathway
• The repair of IDLs between 1-16 bases requires
  Msh2, Msh3 and Pms1 with Mlh2 and Mlh3 playing
  minor roles
• Larger loops around 100 nucleotides involves Msh2
  and Mlh2.
• Transcription of MSH2 MSH6 and MLH1 genes are
  cell cycle regulated reaching peak expression at
  the G1 phase, consistent with their action
  repairing mismatches during S phase.

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Role of PCNA
PCNA interacts with MSH3 and MSH6
PCNA interaction motif QXX(L/I)XXFF
Other residues may also be involved.
Flores-Rozas et al. Nature Genetics, 2000, 26 p
376
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Interaction between PCNA and MSH2-MSH6
Flores-Rozas et al. Nature Genetics, 2000, 26 p
376
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PCNA binds to MSH6
Coprecipitation of Msh2p-Msh6p or
Msh2p-msh6F33F34-AA with PCNA-immobilized on
Affi-Gel beads. After precipitation, the proteins
were fractionated by SDSPAGE and Msh6p was
visualized by western blot. Lane 1, purified
Msh2p-Msh6p lane 2, Msh2p-msh6F33F34-AA lane 3,
Msh2p msh6F33F34- AA bound to the PCNA beads
lane 4, Msh2p-Msh6p lane 5, Msh2p-Msh6p bound to
the PCNA beads lane 6, Msh2p-Msh6p bound to the
mock-treated beads.
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PCNA stimulates MSH2-MSH6 binding to DNA
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MSH2-MSH6 is transferred from PCNA to the mispair
Lau, PJ and Kolodner, RD 2003. JBC Vol. 278, 3,
pp. 1417
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Role of PCNA

• PCNA is naturally loaded onto 3-primer termini
  in replication intermediates and is then left on
  the replicating regions of DNA when replication
  proteins dissociate.
• This could then target MSH2-MSH6 and MSH2-MSH3 to
  replicating regions of DNA where mispaired bases
  would normally be found.
• MSH2-MSH6 associates with PCNA that is tethered
  to DNA to form a nonspecific PCNA-MSH2-MSH6
  complex, and once this complex encounters a
  mispair, the MSH2-MSH6 is transferred from PCNA
  to the mispair, thus activating MMR.
• Such a mechanism would increase the intracellular
  mispair recognition affinity of MSH2-MSH6 at
  newly replicated DNA.

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Strand discrimination

• No GATC methylation in eukaryotes.
• MSH3 and MSH6 have been shown to colocalize with
  the replication foci
• 5 ends of Okazaki fragments, together with PCNA
  which is preferentially deposited on DNA during
  lagging strand replication are used as strand
  discrimination signals by MMR
• Mutations in the leading strand occur 10 times
  more often than on the lagging strand.

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Downstream events role of ATP

• MutS/MutSa binds to ATP after the initial
  mismatch recognition and in complex with
  MutL/MutLa searches along DNA for a strand
  discrimination signal in an ATP hydrolysis
  dependent manner.

Allen, et al The EMBO Journal Vol.16 No.14
pp.44674476, 1997
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Downstream events role of ATP

• Binding of ATP after mismatch recognition
  converts it to a sliding clamp that diffuses
  along the DNA in an ATP hydrolysis independent
  manner with hydrolysis occurring later.

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The excision steps
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Figure 6. Model for Mismatch-, MutS-, MutL-, and
RPA-Dependent Modulation of EXOI Activity EXOI
initiates poorly at a single-strand break, and
MutS facilitates this reaction in a
mismatch-dependent manner. Once EXOI is loaded
by MutS, excision proceeds in a highly processive
manner in the absence of RPA (left diagram). In
the presence of RPA (right diagram), the initial
gap produced by EXOI is filled by the
single-strand binding protein. This results in
displacement of EXOI, MutS, or both proteins
from the helix after removal of about 250
nucleotides. Like a single-strand break, an
RPA-filled gap is a weak substrate for EXOI, but
MutS promotes reloading of the hydrolytic
activity provided that mismatch remains within
the DNA. Hence, in the presence of RPA, excision
is attenuated upon mismatch removal, and this
effect is potentiated by MutS and MutL, which
further suppress EXOI activity on DNA that lacks
a mismatch. Jochen Genschel and
Paul Modrich Molecular Cell, Vol. 12, 10771086,
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Reading

• Mechanism and Functions of DNA mismatch repair
  Cell Research 2008