PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33DNA damage and repair summary 1. Defects in repair cause disease2. Common types of DNA damage3. DNA repair pathwaysDirect enzymatic repairBase excision repair Nucleotide excision repair Mismatch repair Double-strand break repairNon-homologous end joiningHomologous recombinationDNA repair defects cause diseaseCommon types of DNA damage -- 11. Depurination: A, G2. Deamination: C --> U, A --> HypoxanthineCommon types of DNA damage -- 2Pyrimidine dimers (UV induced). Repair pathwaysCommon types of DNA damage -- 3Two carcinogens that mutate (the P53 gene) by base alkylation+ Mismatches (mistakes in DNA synthesis)Interstrand cross-links, Double-strand DNA breaksTotal damage from all mechanisms: 104 - 106 lesions/day!Diverse DNA repair systems • Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes1. Direct repair, e.g. pyrimidine dimers 2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair 5. Double-strand break repair and recombinationBase excision repairBase excision repair pathway (BER).(a) A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone. (b) An AP endonuclease cleaves the phosphodiester backbone near the AP site. (c) DNA polymerase I initiates repair synthesis from the free 3’ OH at the nick, removing a portion of the damaged strand (with its 5’3’ exonuclease activity) and replacing it with undamaged DNA. (d) The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.AP= apurinic or apyrimidinic(a=without)Damaged baseA DNA glycosylase initiates base excision repairExamples of bases cleaved by DNA glycosylases:Uracil (deamination of C)8-oxoG paired with C (oxidation of G)Adenine across from 8-oxoG (misincorporation)Thymine across from G (5-meC deamination)Alkyl-adenine (3-meA, 7-meG, hypoxanthine)Damaged baseHuman alkyl-adenine DNA glycosylaseDNA bent & modified base flipped out of duplex --“Non-Watson-Crick” structureDiverse DNA repair systems • Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes 1. Direct repair, e.g. pyrimidine dimers 2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair 5. Double-strand break repair and recombinationTwo pathways of increasing complexityBase ExcisionrepairNucleotide ExcisionrepairNucleotide excision repair(a) Two excinucleases (excision endonucleases) bind DNA at the site of bulky lesion. (b) One cleaves the 5’ side and the other cleaves the 3’ side of the lesion, and the DNA segment is removed by a helicase. (c) DNA polymerase fills in the gap and (d) DNA ligase seals the nick. UvrA recognizes bulky lesionsUvrB and UvrC make cutsUvrDStructural distortion = signalNucleotide excision repair -- eukaryotesMutations in any of at least seven genes, XP-A through XP-G, cause an inherited sensitivity to UV-induced skin cancer called xeroderma pigmentosum. The XP proteins are among >30 required for nucleotide excision repair.Diverse DNA repair systems • Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes1. Direct repair, e.g. pyrimidine dimers 2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair -- replication errors 5. Double-strand break repair and recombinationMismatch repairMut S binds mismatchMut L links S to HMut H recognizes the parental strandWhich strand is new and which is the parent?Mismatch repairWhich strand is new and which is the parent? The mutation is in the new strand! -CH3 marks the parental strand!MutH - Binds 7-meGATCMutS - Binds mismatchMutL - links MutH and MutSMismatch repair -- RecognitionWhich strand is new and which is the parent? The mutation is in the new strand! A-CH3 marks the parental strand!MutS - Binds mismatchMutL - links MutH and MutSMutH - Binds GmeATCDNA is threaded through the MutS/MutL complex. The complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at a hemimethylated GATC sequence. MutH cleaves the unmethylated strand on the 5’ side of the G in the GATC sequence.Mismatch repair -- Resolution1. The combined action of DNA helicase II, SSB, and one of many different exonucleases (only two are labeled) removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch. 2. The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.Mismatch repair -- Hereditary Non-Polyposis Colon Cancer (HNPCC) gene (Humans)HNPCC results from mutations in genes involved in DNA mismatch repair, including:•several different MutS homologs•Mut L homolog•other proteins: perhaps they play the role of MutH, but not by recognizing hemi-methylated DNA (no 6meA GATC methylation in humans, no dam methylase)Mismatch repair -- MSH proteins -- eukaryotesDefects in mismatch excision repair lead to colon and other cancers.1. MSH2:MSH6 complex binds the mismatch and identifies newly synthesized strand.2. MLH1 endonuclease and other factors such as PMS2 bind, recruiting a helicase and exonuclease, which together remove several nucleotides including the lesion.3. The gap is filled by Pol  and sealed by DNA ligase.Diverse DNA repair systems • Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes1. Direct repair, e.g. pyrimidine dimers 2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair -- replication errors 5. Double-strand break repair and recombinationDouble-strand break repairNO TEMPLATE FOR REPAIR!!Double-strand break repairTwo basic mechanisms: End-joining and Recombination The end-joining pathway of ds break repair is mutagenic, because it removes several base pairs at the break site.Mediated by Ku proteins.Double-strand break repair -- Homologous recombination pathwaysRecBCDRecAC1W1W2C23’5’3’5’3’3’Strand exchange with nicksRecBCD helicase/nuclease in bacteriaRecBCD recognizes ends and unwinds and degrades DNA until it encounters a chi