DNA RepairBiochemistry 302Summary of types of DNA damageSummary of DNA Repair SystemsEnvironmental DNA damage: Reactive chemical agentsDNA repair systems I: Direct change of a modified baseEnvironmental DNA damage: Radiation and pyrimidine dimersDNA repair systems I: Direct repair of thymine dimers by DNA photolyasesDNA repair systems II: Base Excision Repair (BER)Base Excision Repair:Removal of 8-oxo-guanine by hOGG1 (glycosylase/?-lyase)Molecular basis of oxoG recognition(What’s peculiar about these structures?)Multiple enzyme systems ensure that 8-oxoguanine is excluded from DNADNA repair systems III: Nucleotide Excision Repair (NER)Cisplatin-DNA adductsSimilarity of NER repair in humansSite-specific DNA methylation….DNA repair systems IV: Mismatch RepairMechanism of mismatch repair in E. coliExonuclease used depends on location of MutH site relative to mismatchMismatch repair in eukaryotesSummaryDNA RepairBiochemistry 302Bob KelmJanuary 31, 2005Summary of types of DNA damage• Depurination• Deamination• Alkylation• UV photoproducts• Oxidation (maybe most important)– ROS, reactive oxygen species (H2O2, hydroxyl radicals, and superoxide radicals)– ROS generated during irradiation or as byproducts of aerobic metabolism– Defense systems (e.g. catalase and superoxide dismutase)– Oxidants escaping cellular defense can promote…• Deoxyribose oxidation • Base oxidation• Strand breaksSummary of DNA Repair SystemsDNA repair system Types of Damage Enzyme/Proteins involved Direct repair Pyrimidine dimers Alkylated bases Mismatched bases Photoreactivating enzymes Methyl transferasesExcision Repair (nucleotide or base)Alkylated bases Deaminated bases Pyrimidine dimers Lesions that disort the DNA helix DNA glycosylases AP endonuclease UvrABC nuclease (Helicase, DNA Pol I, DNA ligase) Mismatch Repair Mismatched bases on nascent strands Mut proteins Helicases Exonucleases DNA Pol III, ligase Error-Prone Repair Recombinatorial repair Cross-linked bases Intercalation sites Pyrimidine dimers ds breaks UmuC, UmuD RecA RecA and other factorsEnvironmental DNA damage: Reactive chemical agents• Direct action vs damage cause by metabolic by-products• Precursors of nitrous acid (HNO2) e.g. NaNO2, NaNO3, and nitrosamine– Accelerate deamination– Food preservatives• Alkylating agents– Replace H atom– Methylation of purines results in altered base-pairing (O6-methylguanine cannot pair with cytosine)– Used experimentally as DNA modifying agents (mutagens)dimethylsulfateDNA repair systems I: Direct change of a modified base• O6-Alkylguanine alkyl-transferase (methyltransferase)– Catalyzes transfer of alkyl to Cys residue resulting in protein turnover – Alkylated form self-regulates its own transcription– Energetically costly- an entire protein consume to fix 1 base• MutT nucleotide hydrolase– Accumulates in O2-stressed cells (8-oxo-G can bp w/A)– Cleaves 8-oxo-dGTP prior to incorporation in DNA• Photoreactivation86Fig. 25.11Environmental DNA damage: Radiation and pyrimidine dimers• One of the first forms of DNA damage discovered • Irradiation of bacteria w/ 260 nm light → condensation of adjacent ethylene groups • Human skin cells particularly susceptible• Ionizing radiation (x-rays and gamma rays)– Ring opening and base fragmentation– Breaks in DNA backbone• UV + ionizing radiation exposure accounts ~10% of all DNA damage caused by environmental agents lethal mutagenic AT→GC200-400 nm near-UVLehninger Principles of Biochemistry, 4th ed., Ch 8DNA repair systems I: Direct repair of thymine dimers by DNA photolyasesFig. 25.1056• Enzyme (E. coli and yeast) uses a “photosynthesis-like” free-radical-dependent mechanism.• Enzyme binds to lesion in the dark and breaks C5-C5 and C6-C6 bonds in the light.• Enzyme contains two cofactor chromophores that absorb light at specific λs (photoantennaMTHFpolyGlu transmits light energy to FADH−) .• Excited FADH•transfers an electron to the dimer. Electronic rearrangement restores thymine monomers.• Enzyme is not found in mammalian cells.DNA repair systems II: Base Excision Repair (BER)• Damaged bases repaired – Products of deamination (uracil), depurination, alkylation, and oxidation– Thymine dimers (phage T4 specific mechanism)• Four-step mechanism– Removal of damaged base via specific DNA-N-glycosylase– Nicking of abasic strand by AP endonuclease– Gap repair synthesis by DNA polymerase I – Nick repair by DNA ligase• AP endonucleases types– Cut 5′ to abasic site– Cut 3′ to abasic siteLehninger Principles of Biochemistry, 4th ed., Ch 25Base Excision Repair:Removal of 8-oxo-guanine by hOGG1 (glycosylase/β-lyase)• OGG1 recognizes oxoG opposite C. • Active site Lys of hOGG1 attacks the C1′ of deoxyribose resulting in the extrusion of oxoG.• BER apparatus restores correct G/C base-pairing.Hoogsteen modeAminal intermediate Schiff baseεrearrangementDNA backbone cleavageFrom Bruner, S. D. et al. Nature 403: 859 (2000)(Example of a modified base-specific DNA glycosylase)Molecular basis of oxoG recognition(What’s peculiar about these structures?)From Bruner, S. D. et al. Nature 403: 859 (2000)Multiple enzyme systems ensure that 8-oxoguanine is excluded from DNAMechanisms to prevent GC→AT or AT→GC conversions depending upon route of entry (BER, base excision repair)•MutM = E.coli analog of hOGG1 •MutT = 8-oxo-dGTP nucleotide hydrolase•MutY = adenine DNA glycosylase (only) works for adenine opposite oxoG•Redundancy built into the system.Next round correctionFig. 25.14DNA repair systems III: Nucleotide Excision Repair (NER)• Typically occurs with bulky lesions that distort the DNA helix– pyrimidine dimers not removed by BER– Intra-strand G crosslinks (cisplatin-induced)– alkylation• E. coli machinery (similar in yeast and mammals)– UvrA, B, C excinuclease (catalyzes two specific endonucleolytic cleavages)– UvrD helicase II– DNA polymerase and DNA ligasec) Monofunctional adducts ~ 2%a) Intra-strand crosslink ~ 90%b) Inter-strand crosslink ~ 5%http://www.md.huji.ac.il/courses/bioorganic/cisplatin_3.ppt Cisplatin = cis-diaminedichloroplatinum (cancer chemotherapeutic agent but highly toxic, only short-term efficacy)Cisplatin-DNA adductsNote how guanines become de-stacked.H-bonding between N7 of G & cisplatinStep 1:Dimeric UvrA binds UvrB and tracks along DNA.Step 2: UvrAB complex sees lesion and