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UVM BIOC 302 - DNA Replication III & DNA Damage/Modification

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DNA Replication III& DNA Damage/ModificationBiochemistry 302January 27, 2006Bacterial replication organized into a membrane-bound replication “factory”Lehninger Principles of Biochemistry, 4th ed., Ch 25This mechanism facilitates chromosome partitioning into daughter cells.Two replisomes do not travel away from one another but rather are linked to each other.Features of DNA replication in eukaryotes• Many origins > 5,000/genome which only initiate once per cell cycle• Ori: less sequence conservation• Origin Recognition Complex (ORC)– Six polypeptides, binds to yeast ARS in ATP-dependent manner & recruits MCM helicase complex– Role for phosphorylation, DNA methylation, and histone remodeling• Mitochondrial DNA– Fixed origins but use two unidirectional forks (~1 h to complete replication) Fig. 28.18Fig. 24.39from Lodish et al. Molecular Cell Biology, 3rdEdition16 kb circular duplexLicensing a site for initiation of DNA replication (E. coli vs budding yeast)Robinson, N.P. and Bell, S.D. (2005) FEBS J. 272: 3757-3766ArchealWith the exception of fission yeast S. pombe, all eukaryotic ORCs characterized to date require ATP to bind DNA.Eukaryotic Origins: no obvious sequence signature, noncodingregions, AT-richness, asymmetric strand compositionKinetic features/enzymology of eukaryotic DNA replication• Slower fork movement but larger number of origins (~103 per 108bp chromosome)• Five different polymerases (distinguished by intracellular location, kinetic properties, and response to inhibitors): – Pol α - Okazaki fragment primer synthesis. (Primase activity but no 3′→5′ exonuclease activity and not very processive). – Pol δ - Major leading and lagging strand polymerase w/ 3′→5′ exonuclease activity. Highly processive w/ bound PCNA (analogous to E. coli Pol III).– Pol ε - DNA repair and Okazaki fragment RNA primer removal (perhaps, not definitive). – Pol β and Pol γ - repair and mitochondrial DNA replication.Properties of eukaryotic DNA polymerasesPCNA ~ E. coli β sliding clamp of Pol III holoenzymeRFC ~ E. coli clamp loader γ complex of Pol III holoenzymeRFA ~ E. coli SSB; RNA primer removal by FEN-1/RNaseH1 or Pol εTable 24.3Nuclear genomes & linear chromosomes: nucleosome replication & gaps on 5′ endsFig. 24.40Fig. 28.17Replicating and protecting the ends of linear chromosomesT-loop formation: telomere repeat binding factorsEM mouse hepatocyte chromosome T-loopLehninger Principles of Biochemistry, 4th ed., Ch 26Mechanisms for ensuring fidelity of DNA replication• Metabolic: balanced levels of dNTPs• Structural 1: complementary base pairing between dNTP and template (Error ~1 in 103 to 104bp/round of replication)• Structural 2: induced fit between Pol and DNA (Cumulative Error ~1 in 105 to 106)• Enzymatic 1: proofreading by 3′ to 5′exonuclease (Cumulative Error ~1 in 107 to 108)• Enzymatic 2: mismatch and other repair systems (1 in 1010bp/generation)Overview of DNA RestructuringRestriction andModification(protective mechanisms in prokaryotes, useful in recombinant DNA techniques)Recombination (redistribution of genomic contents, reproduction, repair)Repair (in response to DNA damage)Transposition and Amplification(developmental processes and/or responses to external stressFig. 25.1Consequences of DNA damage…..Cancer AgingMutationsReplication ErrorsPersistent DamageGenomic InstabilityDNA DamageDNA ReplicationDNA RepairXMechanisms ensuring that information content is transmitted w/o error• High accuracy replication and editing – 3′ exonuclease-mediated proofreading– Uracil DNA glycosylase (1 in E. coli, 4 types in humans) • Mis-incorporation of dUMP (rare)• Cytosine deamination (common occurrence)• Associated w/ the replisome (human UNG)• Repairing damaged DNA arising from….– Replicative errors not corrected by proofreading activity of DNA polymerases– Spontaneous alterations in covalent structure of nucleotide (very slow but physiologically significant, permanent change = mutation)– Environmental damage (chemical or photochemical)Spontaneous non-enzymatic reactions of nucleotides: deamination• Loss of exocyclic amino group• C → U (1 out of every 107cytidine residues/24 h) so ~100 events/day in a mammalian cell. Rate higher in ssDNA.• Oxidative deamination of adenine and guanine occurs at ~ 1/100 the rate of cytosine deamination.• What is the potential genetic consequence of such an event?462DNA Damage: Chemical structure of bases that are mutagenic (i.e. produce altered, non-Watson-Crick base-pairs)Deamination Base analogs: used experimentally as mutagens bp w/T & Gbp w/T & C7931Spontaneous non-enzymatic reactions of nucleotides: depurination• Hydrolysis of the N-β-glycosyl bond• Higher rate for purines than pyrimidines (1 out of every 105guanosine residues or 104 events/24 h in mammalian cell) • Slower in ribonucleotides and RNA (probably not physiologically significant)• Accelerated by low pH ~3 resulting in apurinic acid → ring opening, linear aldehyde configurationEnvironmental 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 8Environmental 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)dimethylsulfateSummary of types of DNA damage• UV photoproducts• Depurination• Deamination• Alkylation• 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…•


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UVM BIOC 302 - DNA Replication III & DNA Damage/Modification

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