DNA Damage and Repair

1
DNA Damage and Repair
2
Human Syndromes with Defective Genome Maintenance
Affected Mechanism
Cancer Predisposition
Syndrome
Xeroderma pigmentosum
NER/TCR
UV-induced skin cancer
Cockayne syndrome
NER
None
Trichothiodystrophy
NER/TCR
None
Ataxia telangectasia
DSB repair
Lymphomas
Nijmegen breakage syndrome
DSB repair
Lymphomas
BRCA1/BRCA2
Homologous recombination
Breast cancer
Werner syndrome
Homologous recombination
Various cancers
Bloom syndrome
Homologous recombination
Lymphomas
HNPCC
Mismatch repair
Colorectal/ovarian
3
Colorectal Tumors
Familial Adenomatous Polyposis
Hereditary Nonpolyposis Colorectal Cancer
4
Colorectal Tumor Progression
Normal Crypt Cells
Hyperplastic Cells
Dysplastic Cells
5
HNPCC and Mismatch Repair
Clues to involvement of mismatch repair in HNPCC
Linkage analysis - Individual families showed
tight linkage of HNPCC to two loci Microsatellite
markers used for loss of heterozygosity in
tumors showed alleles not present in normal
tissues Microsatellite repeats unstable in HNPCC
were equally unstable in yeast carrying mismatch
repair mutations
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Prevalence of Colorectal Cancers
7
HNPCC Are Deficient in Mismatch Repair

• HNPCC cell extracts defective in mismatch repair
• Vast majority of HNPCC exhibit microsatellite
  instability
• 50 of HNPCC kindred families harbor mutations
  in either hMSH2 or hMLH1 a few of the others
  carry mutations in hPMS1, hPMS2, MSH6 or MLH3

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E. coli Mismatch Repair
MutS
CH3
CH3
3
5
5
3
9
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
10
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
11
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
12
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
13
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
14
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
15
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
16
E. coli Mismatch Repair
MutS
CH3
CH3
MutL
3
5
5
3
17
E. coli Mismatch Repair
CH3
CH3
3
5
5
3
18
E. coli Mismatch Repair
CH3
CH3
3
5
5
3
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E. coli Mismatch Repair - Bidirectional
CH3
CH3
5
3
3
5
CH3
CH3
CH3
CH3
5
3
5
3
3
5
3
5
exoVII or RecI
exoI
CH3
CH3
CH3
CH3
5
3
5
3
3
5
3
5
polIII SSB
polIII SSB
CH3
CH3
CH3
CH3
5
3
5
3
3
5
3
5
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Structure of the E. coli MutS Protein
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Yeast Mismatch Repair Genes
mutS homologs
mutL homologs
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Phenotypes of MMR Mutants

• Increased mutation rate
• Microsatellite Instability
• Resistance to killing by alkylating agents

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Assay for Mismatch Repair
XhoI HindIII
CTCGAGAGCTT CAGCTTTCGAA
Correct lower strand
Correct upper strand
No Correction
HindIII site
XhoI site
No cutting
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Assay for Mismatch Repair
Correct upper strand
Lac Lac-
CTGACTC GACCGAG
No Correction
Correct lower strand
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Initiation of Microsatellite Instability
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Initiation of Microsatellite Instability
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Mutation Rates in MMR Mutants
Forward Mutation Rate to CanR
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Effect of Microsatellite Repeat Unit Length and
Genetic Background on MIN
Microsatellite Repeat Length
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Summary of MSH Mutant Properties

• msh6 effect gtgt msh3 effect on mutation rate
• msh3 effect gtgt msh6 effect on MIN
• msh3 synergistic with msh6 in both
• msh2 gtgt msh3 or msh6 in both
• msh2 not synergistic with either msh3 or msh6
• msh2 effects msh3 msh6 effects

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Model for Msh Functions
Mispair Repair
Msh2
Msh3
2-8 Base Insert Repair
1 Base Insert Repair
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Other Proteins Involved in Mismatch Repair

• MutL Homologs MLH1, PMS1, MLH3
• Mlh1-Pms1 complex required for most mismatch
  repair
• Mlh1-Mlh3 complex required for some
  insertion/deletion mispairs
• Exonucleases
• ExoI 5-gt 3 exonuclease weak mutator
  phenotype
• Fen1 (Rad27) 5 -gt 3 exonuclease weak mutator
  phenotype
• PCNA

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Pathways for Mismatch Repair in Human Cells
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Phenotypes of Mice with MMR Mutations
Later onset than in msh2 mice
Lymphomas, skin and others
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Mouse Models of HNPCC
Msh6-/- mice show defects in mismatch repair
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Mouse Models of HNPCC
Msh6-/- mice show reduced survival due to
increased tumor incidence
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Mouse Models of HNPCC
Tumors observed in Msh6-/- mice
Adenocarcinoma stained with anti-APC
Tumor of the small intestine
Lymphoma stained with B-cell Ab
Mesenteric lymphoma
Hepatoma
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What Dictates Strand Specificity?

• DNA Methylation?
• dam methylion in E. coli
• CpG methylation in mammals
• DNA Nicks?
• PCNA?

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What Dictates Strand Specificity?
PCNA Colocalizes with MSH3 and MSH6 at DNA
Replication Nodes in Cells
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Distribution of Human HNPCC Mutations
MMR Gene
of Mutations
MSH2
125 (38)
MLH1
164 (49)
PMS1
1 (0.3)
PMS2
5 (2)
MSH6
30 (9)
MLH3
7 (2)
International Collaborative Group on HNPCC (2001)
But, these account for only 70 of all HNPCC
families
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Epigenetic Contribution to Sporadic CRC

• In a study of 46 sporatic MSI colorectal tumors
• 36 (78) had reduced MLH1 protein, of which
• 83 had MLH1 promoter hypermethylation
• 24 had MLH1 LOH
• 13 had MLH1 somatic mutations
• 7 (15) had reduced MSH2 protein, of which
• None exhibited MSH2 hypermethylation
• 29 had MSH2 LOH or mutation

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Are Some Genes Hypermutable in MSI Tumors?

• TGFb-IIR
• Tumor suppressor gene
• A10 track in the 5 region of the coding
  sequence
• High incidence of frameshift mutants in MSI
  tumors
• Other tumor suppressor genes with internal
  nucleotide repeats
• IGFIIR growth factor receptor
• E2F4 cell cycle transcription factor
• Bax, Apaf1 pro-apoptotic factors
• TCF4 Wnt regulated transcription factor

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MMR and Apoptotic Signaling

• MMR- cells (bacterial, yeast, human) are
  resistant to alkylating agents
• Msh2-/- primary cells are resistant to MNNG
  induced apoptosis
• Mlh1 or Msh2 overexpression induces apoptosis
• p63 and p73 are required for DNA damage induced
  apoptosis
• cisplatin induces p73 in MMR but not MMR- cells