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Mismatch Repair

Mismatch-when conventional but noncomplementaryW-C bp occur in the double helix

8 classes:G-T, A-C pairs, cause transition mutationsG-G,A-A,T-T,C-C, C-T,G-A,cause transversion miut.

Insertion or deletion mutations are also recognized

Source- heteroduplexes formed during replication orrecombination

Transform with heteroduplex, found it was corrected

Wagner and Meselson-1976

Mismatches are subject to a repair system that dis-criminates newly synthesized from parental strands

Selective elimination of errors from newly synthesized DNA

Proposed the selectivity might be based on methylation

Methylation is a post-synthetic event, thus newly synthesized DNA is transiently hemi-methylated

N6-methyl adenine - dam methylase (GATC/CTAG)m5C-dcm methylase

dam mutants-hypermutable, overproducers also hypermutable

Miroslav Radman

Direct evidence - transform with heteroduplex DNA con-taining mismatched bases or small deletions and insertion

Mutants defective in correction in E. coli are:

mutH- binds hemi-methylated DNA, latent endonuclease

mutL-mediator, activates latent endo of mutH

mutS-recognize mismatch

xHemi-methylated Fully methylated Unmethylated

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Mechanism

Paul Modrich, in vitro system- a cell free system that supportsmethyl directed reaction requiring mutH, mutL, and mutS

Strand specific process involving excision of long tracts, several kb

Adenine methylation d(GATC) modulates strandedness

Correction occurs on the unmethylated strand

Recognize mismatch, 97 kDa, AAA+ ATPase,promote loopBy sliding

Recognize GATC25 kDa

Latent endo

Cleaves unmethyla-ted strand-stimulatedby mismatch, mutL,and mutS

mutS

mutH

mutL

Architectural role70 kDa dimer

Excision reaction

Bidirectional - Either a 5’ to 3’ or a 3’ to 5’ nuclease, depending on which methyl group directs incision

Exonuclease. i.e. ssDNA end required

Therefore, a helicase involved, uvrD

Resynthesis reactionPol III

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1993  Bert Vogelstein-two papers

1)  Simple repeats are unstable in a set of sporadic colon cancer tumors

Deletions of up to 4 NT in poly(dA) tracts

Insertions and deletions in microsatellite sequences of di-or trinucleotide repeats

2)  HNPCC-hereditary, nonpolyposis colorectal cancer- One of the most common cancer predisposition syndromesdevelop cancer before age 5020% of colon cancers, 2nd largest killer behind lung cancer

Role of heredity is established and 2 loci on chromosome 2had been linked to HNPCC in genetic studies

In studying the inheritance of the autosomal dominant marker,found that tumors developing in these individuals had alterations in their microsatellite sequences

They called this RER+, replication error phenotype

He dug in to clone this gene-positional cloning

One of the papers was published in the NYT

Paul Modrich noticed the similarity to mismatch repair in bacteria and to some yeast work (from Tom Petes)

In yeast, responsible for expansion and contraction of simple repeat sequences

Poly(dGT)10-30

Several human inherited disorders, fragile X syndrome,myotonic dystrophy, Huntington’s disease are associatedwith expansions of triplet repeats

Several other cancers are associated with alterations in dinucleotide repeat tracts

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Collaborated to look for mut homologs in the region, which theypredicted should be affected by HNPCC mutations, and to show cell lines were defective in MMR

Collaborated to look for mut homologs in the region, which they predicted should be affected by HNPCC mutations, and to show cell lines were defective in MMR

hMSH2 (MutS Homolog) was found in the regionEvidence it was causative:

-  in HNPCC patients, hMSH2 genes were mutated

-MSH2 segregates with HNPCC and is mutated in many sporadiccolon cancers

-Modrich showed that RER+ cells were defective in mismatch repair in vitro

- invented an assay similar to E. coli, strand specific MMR system dependent on MSH2- early step defective in RER+ cells

-3 additional genes, homologs of mutL (MLH) identified in otherHNPCC families

humanMutSα•heteroduplexcompleximagecourtesyofLorenaBeese

MechanismsinE.coli&humanmismatchrepair

PaulModrichHHMI&DeptBiochemistry

DukeUniversity

Modrich, P. J. Biol. Chem. 2006;281:30305-30309

Substrates and requirements for in vitro mismatch repair

5’ 3’ substrate

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Modrich, P. J. Biol. Chem. 2006;281:30305-30309

5' to 3' default hydrolytic system removes mismatch

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Conclusion:

Most theories of cancer attribute cancer to somatic andinherited genetic changes that lead to alterationsIn GROWTH CONTROL (tumor suppressor, oncogene)

Progressive somatic mutations are associated with several cancers

It has been postulated that an early step might confera mutator phenotype that leads to intrinsic genomicInstability (MMR mutants increase mutation rate 100-1000x)

Caveat: in yeast, mutations in mismatch repair genes led to increase in telomere maintenance by recombination - i.e. MMR tumors don’t need to reactivatetelomerase in order for tumor cells to proliferate

Gene rearrangement in cancer could be due to promotion ofillegitimate recombination between quasi-homologous sequences by MMR

Salmerichia - Salmonella and E. coli 5% apart but in mutmutants, they can recombine

Fischl et al. Cell 75, 10207-10038 (1993) KolodnerLeach et al. Cell 75, 1215-1225 (1993) VogelsteinParsons et al. Cell 75, 1227-1236 (1993) Modrich

DNA DAMAGE TOLERANCE Stalled Replication forks Upon replication fork stalling, PCNA is ubiquitinated to promote DNA damage toler

Chang and Cimprich, Nature Chem Bio 5,82 (2009)

Regulation of Post-replication Repair by PCNA Modification

Pathway choice is important: different modifications for different types of damage

PCNA

Chang and Cimprich, Nature Chem Bio 5,82 (2009)

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DNA Damage Tolerance at Stalled Replication Forks: It’s OK to make mistakes

Chang and Cimprich (2009)

Chang and Cimprich, Nature Chem Bio 5,82 (2009)

Simultaneous TLS repair and Checkpoint Activation- PCNA And 911

Chang and Cimprich, Nature Chem Bio 5,82 (2009)

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d.

e.

f.

Translesion synthesis, Removal of unhooked X-link

DNA2 5’ to 3’ resection

RAD51 strand invasion

HR

a.

b.

c.

DNA replication

FancD2/FancI, Endonuclease,Unhooking of X-link

Interstrand crosslink Repair – The Fanconi anemia pathway

Long, Walter (2011)