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DNA RepairBiochemistry 302January 30, 2006Summary 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)dimethylsulfateMutagenic: O6-methylguanine can base-pair with thymineDNA repair systems I: Direct change of a modified base• O6-Alkylguanine alkyltransferase(methyltransferase)– Catalyzes transfer of alkyl group to Cysresidue resulting in protein turnover – Alkylated form self-regulates its own transcription– Energetically costly: an entire protein consumed to fix 1 base • AlkB protein (α-ketoglutarate-Fe2+-dependent dioxygenase)– Catalyzes oxidative demethylation of alkylated ring amines of A (N1) & C (N3)– Methylation can occur in DNA when single-stranded and this affects base-pairing• MutT nucleotide hydrolase– Not used for DNA repair per se– Accumulates in O2-stressed cells (8-oxo-G can bp w/A)– Cleaves 8-oxo-dGTP so it cannot be incorporated into DNA (8-oxo-dGTP → 8-oxo-dGMP + PPi)• Photoreactivation (unique to E. coli and lower eukaryotes)86Fig. 25.11Fe2+-dependent oxidative demethylationcatalyzed by E. coli AlkBP. O. Falnes, R. F. Johansen and E. Seeberg (2002) Nature 419, 178-182Note how demethylation is coupled to decarboxylation of α-KG. Human homologs: ABH2 and ABH3DNA repair systems I: Direct repair of thymine dimers by DNA photolyases• 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.Fig. 25.1056DNA 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 25Summary of types of DNA damage• Depurination• Deamination• Alkylation (chemotherapeutic agents)• UV photoproducts • Oxidation (maybe most pervasive)– 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 particularly guanine• Strand breaksBase Excision Repair:Removal of 8-oxo-guanine by hOGG1 (glycosylase/β-lyase)From Bruner, S. D. et al. Nature 403: 859 (2000)• 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.(Example of a modified base-specific DNA N-glycosylase)Hoogsteen modeAminal intermediate Schiff baseεrearrangementDNA backbone cleavageMolecular basis of oxoG:C recognition(What’s peculiar about these structures?)From Bruner, S. D. et al. Nature 403: 859 (2000)hOGG1 is a member of the HhH (helix hinge helix: αK:hinge:αL) superfamily.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 DNA repair systems.Next round correctionFig. 25.14DNA repair systems III: Nucleotide Excision Repair (NER)• Typically occurs with bulky lesions that distort the DNA helix– pyrimidine (thymine) dimersHuman cells: no photolyase– 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 ligasea) Intra-strand crosslink ~ 90%b) Inter-strand crosslink ~ 5%c) Monofunctional adducts ~ 2%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 with energy provided by ATP hydrolysis bends DNA.Step 3: UvrA dissociates and UvrC associates with UvrB.Steps 4 & 5: UvrBC complex cuts on both sides of the lesion (UvrB nicks at the 5thPDE bond on 3′ side, UvrCat the 8thPDE bond on the 5′ side).Step 6: UvrD (helicase II) removes 12-13 nt fragment. DNA polymerase I fills in missing piece.Step 7: Nick sealed by DNA ligaseExcision repair by E. coli UvrABC excinuclease systemFig. 25-12Similarity of NER repair in humans• Human excinuclease– 16 polypeptides: no homology to UvrABC subunits– NER sole repair pathway for pyrimidine dimers in humans• Xeroderma pigmentosum (XP)– Deficiency in one or more XP NER proteins (XPA→XPG) – Extreme sensitivity


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