Nucleic Acid ModificationBiochemistry 302Overview of DNA RestructuringConsequences of DNA damage…..Mechanisms ensuring that information content is transmitted w/o errorSpontaneous non-enzymatic reactions of nucleotides: deaminationSpontaneous non-enzymatic reactions of nucleotides: depurinationEnvironmental DNA damage: Radiation and pyrimidine dimersEnvironmental DNA damage: Reactive chemical agentsSummary of types of DNA damageSite-specific DNA methylation….….and other nucleic acid-specific modificationsNucleic Acid ModificationBiochemistry 302Bob KelmJanuary 28, 2005Overview 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…..DNA DamageXDNA RepairDNA ReplicationCancer AgingMutationsReplication ErrorsPersistent DamageGenomic InstabilityMechanisms 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.• Deamination of adenine and guanine occurs at ~ 1/100 the rate of cytosine deamination.• What is the potential genetic consequence of such an event?Spontaneous 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…• Deoxyribose oxidation • Base oxidation• Strand breaksSite-specific DNA methylation…. • Bacteria– Restriction & modification– Mismatch error correction• Eukaryotes– Tissue-specific inactivation of genes during development – Suppression of transposon migration– Formation of Z-DNA• Occurrence– E. coli: N6of adenine (major), N4of cytosine (minor), GATC restriction sites– Eukaryotes: 3-5% of cytosines (C5), mainly in CpG islands (animal cells and higher plants, absent in insects)….and other nucleic acid-specific modificationsThese are found in tRNA.These are found in