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DNA Damage and Repair

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Ataxia telangectasia. DSB repair. Lymphomas. Nijmegen breakage syndrome. DSB repair. Lymphomas ... Linkage analysis - Individual families showed tight linkage ... – PowerPoint PPT presentation

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Title: 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
6
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

8
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
19
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
20
Structure of the E. coli MutS Protein
21
Yeast Mismatch Repair Genes
mutS homologs
mutL homologs
22
Phenotypes of MMR Mutants
  • Increased mutation rate
  • Microsatellite Instability
  • Resistance to killing by alkylating agents

23
Assay for Mismatch Repair
XhoI HindIII
CTCGAGAGCTT CAGCTTTCGAA
Correct lower strand
Correct upper strand
No Correction
HindIII site
XhoI site
No cutting
24
Assay for Mismatch Repair
Correct upper strand
Lac Lac-
CTGACTC GACCGAG
No Correction
Correct lower strand
25
Initiation of Microsatellite Instability
26
Initiation of Microsatellite Instability
27
Mutation Rates in MMR Mutants
Forward Mutation Rate to CanR
28
Effect of Microsatellite Repeat Unit Length and
Genetic Background on MIN
Microsatellite Repeat Length
29
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

30
Model for Msh Functions
Mispair Repair
Msh2
Msh3
2-8 Base Insert Repair
1 Base Insert Repair
31
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

32
Pathways for Mismatch Repair in Human Cells
33
Phenotypes of Mice with MMR Mutations
Later onset than in msh2 mice
Lymphomas, skin and others
34
Mouse Models of HNPCC
Msh6-/- mice show defects in mismatch repair
35
Mouse Models of HNPCC
Msh6-/- mice show reduced survival due to
increased tumor incidence
36
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
37
What Dictates Strand Specificity?
  • DNA Methylation?
  • dam methylion in E. coli
  • CpG methylation in mammals
  • DNA Nicks?
  • PCNA?

38
What Dictates Strand Specificity?
PCNA Colocalizes with MSH3 and MSH6 at DNA
Replication Nodes in Cells
39
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
40
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

41
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

42
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
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