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Chapter 5

Chapter 5. DNA Repair. TABLE 5-2: Inherited Syndromes with Defects in DNA Repair NAME PHENOTYPE ENZYME OR PROCESS AFFECTED HNPCC colon cancer mismatch repair MSH2, 3, 6, MLH1, PMS2

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Chapter 5

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  1. Chapter 5 • DNA Repair

  2. TABLE 5-2: Inherited Syndromes with Defects in DNA Repair NAME PHENOTYPE ENZYME OR PROCESS AFFECTED HNPCC colon cancer mismatch repair MSH2, 3, 6, MLH1, PMS2 Xeroderma pigmentosum (XP) skin cancer, cellular UV sensitivity, nucleotide excision-rep groups A-G neurological abnormalities XP variant cellular UV sensitivity translesion synthesis by DNA polymerase  Ataxia-telangiectasia (AT) leukemia, lymphoma, cellular -ray ATM protein, a protein kinase activated sensitivity, genome instability by double-strand breaks BRCA-2 breast and ovarian cancer repair by homologous recombination Werner syndrome premature aging, cancers accessory 3’-exonuclease and DNA at several sites, genome instability helicase Bloom syndrome cancer at several sites, accessory DNA helicase for replication stunted growth, genome instability Fanconi anemia groups A-G congenital abnormalities, leukemia, DNA interstrand cross-link repair genome instability 46 BR patient hypersensitivity to DNA-damaging DNA ligase I agents, genome instability

  3. Spontaneous mutations that require DNA repair

  4. Sites of DNA base damage

  5. DNA intercalating agents

  6. Depurination and deamination

  7. Pyrimidine dimers

  8. How do chemical modifications cause mutations?

  9. Mutations caused by DNA modifications

  10. General strategies for DNA repair • Photolyases • Alkyltransferases Direct Reversal of DNA damage: Excision-Repair: • Base Excision Repair • Nucleotide Excision Repair (NER) • Mismatch repair (MMR) • Transcription-coupled Repair (NER) • Non-homologous end-joining (NHEJ) • Homologous recombination (HEJ) Double-Strand Break Repair: Error-prone Repair: • Trans-lesion replication (TLS) (SOS)

  11. DNA photolyase

  12. DNA methyltransferates

  13. Glycosylases use base flipping to identify modified bases

  14. Glycosylases use base flipping to identify modified bases

  15. Nucleotide deamination

  16. The cell can use multiple mechanisms to remove damaged bases

  17. DNA mismatch repair

  18. DAM methylation

  19. Removal of mismatched DNA

  20. Bacterial Nucleotide Excision Repair

  21. RNAP Transcription-Coupled NER DNA lesion Recognition of Stalled RNA Pol. Recruitment of NER factors Removal of 24 to 32 bp oligonucleotide Fill-in of Gap

  22. Methods for repairing double stranded breaks Rad51

  23. repaired plasmid gapped plasmid Damaged molecule gains information from the donor chromosome chromosome DSB -> homologous recombination in mitosis Formulated to account for several experimental observations in budding yeast… DNA transformation experiments • Linearized plasmids, cut in a region of similarity (“homology”) with a chromosome, transformed into yeast are repaired and integrated into the chromosome efficiently • Plasmids containing a gap in the homologous region are repaired by filling-in of all of the information present on the template but missing on the plasmid

  24. Non-homologous End Joining (NHEJ) KU: 2 proteins (Ku70p/Ku80p) Recognizes ends DNA-dependent protein kinase (DNA-PKCS) Rad50p-Mre11p-NBS1p: exonuclease complex to process ends for ligation; checkpoint signaling Ligase IV rejoins ends

  25. Non-homologous End Joining (NHEJ) One function of the MRE11/RAD50/NBS1 (MRN) complex may be to bring the ends together (Xsr2) Rad50 has structure similar to SMC proteins

  26. Human syndromes associated with defects in DSB repair Ataxia telangiectasia Nijmegen breakage syndrome Breast and Ovarian Cancer (BRCA1-2)

  27. General strategies for DNA repair • Photolyases • Alkyltransferases Direct Reversal of DNA damage: Excision-Repair: • Base Excision Repair • Nucleotide Excision Repair (NER) • Mismatch repair (MMR) • Transcription-coupled Repair (NER) • Non-homologous end-joining (NHEJ) • Homologous recombination (HEJ) Double-Strand Break Repair: Error-prone Repair: • Trans-lesion replication (TLS) (SOS)

  28. E. coli DNA polymerases Enzyme Gene Function I polA repair II polB replication start III polC replicase IV dinB translesion replication V umuD’2C translesion replication

  29. DNA pol Function Structure •  (alpha) Nuclear rep / primer syn 350 kD tetramer •  (delta) Nuclear replication / BER 250 kD tetramer •  (epsilon) Nuclear replication / BER 350 kD tetramer •  (gamma) Mitochondrial replication 200 kD dimer • high fidelity repair •  (beta) Base excision repair 39 kD monomer • low fidelity repair •  (zeta) Thymine dimer bypass heteromer • (eta) Base damage repair monomer •  (iota) Meiosis/ TLS / somatic hyper monomer • (kappa) Deletion and base substitution monomer  (theta) DNA repair of crosslinks monomer  (lambda) Meiosis-assoc DNA repair monomer  (mu) Somatic hypermutation monomer Rev1 Translesion synthesis monomer

  30. Specialized DNA polymerases Have error rate 2-4 orders of magnitude higher than replicative DNA polymerases on undamaged templates Lack 3’-5’ proofreading exonuclease activity Not very processive They support TLS of damaged DNA Induced in bacteria; constitutively expressed in eukaryotes

  31. Translesion synthesis DNA Gene infidelity on undamaged DNA (relative to pol  = ~1) POLB ~50 REV3L ~70 POLK ~580 POLH ~2000 POLI ~20,000 POLL ? POLM ? POLQ ? Rev1 REV1L ? Errol C. Friedberg, Robert Wagner, Miroslav Radman Specialized DNA Polymerases, Cellular Survival, and the Genesis of Mutations SCIENCE VOL 296 31 MAY 2002

  32. Translesion synthesis TLS M N Primer Extension M N Resumption of Replication M N Repair of Lesion M N N M Errol C. Friedberg, Robert Wagner, Miroslav Radman Specialized DNA Polymerases, Cellular Survival, and the Genesis of Mutations SCIENCE VOL 296 31 MAY 2002

  33. } TABLE 1 Lesion bypass by one or two DNA polymerases Lesion bypass by Two Pols Error-free or DNA lesion One Pol Inserter Extender mutagenic 8-oxoG Polη Error-free CPDs at TT, TC, CC sites Polη Error-free (6-4) photoproducts: at TT Polη Polζ Mutagenic at TC and CC Polη Polζ Error-free at TT, TC, and CC Polι Polζ Mutagenic Abasic sites Polδ Polζ Mutagenic Polη Rev1 Polι Tg Polδ Polζ Error-free AAF- or AF-adducted G Polη Polζ Error-free γ -HOPdG Rev1 Polζ Error-free Polι Polκ Error-free CPDs = cyclobutane pyrimidine dimers 6-4 = another UV photo damage Tg = Thymine glycol N-2-acetyl aminofluorene (AAF) γ -hydroxy-1,N2-propano-deoxyguanosine (γ -HOPdG) ( Satya Prakash, Robert E. Johnson, and Louise Prakash EUKARYOTIC TRANSLESION SYNTHESIS DNA POLYMERASES: Specificity of Structure and Function Annu. Rev. Biochem. 2005. 74:317–53

  34. Eukaryotic DNA polymerases

  35. MicroReview Evolution of mutation rates in bacteria. E. Denamur and I. Matic Molecular Microbiology (2006) 60(4), 820–827 Fig. 3. Diffusion test for antibiotic susceptibility of E. coli natural isolates using commercial fosfomycin discs. The mutator strain (natural mutS– mutant), which generates mutations conferring resistance to the antibiotic at a high rate, is clearly differentiated from the nonmutator natural isolate by the presence of squatter colonies inside growth inhibition zone.

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