title: Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase.
abstract: Genomic DNA is prone to oxidation by reactive oxygen species. A major product of DNA oxidation is the miscoding base 8-oxoguanine (8-oxoG). The mutagenic effects of 8-oxoG in mammalian cells are prevented by a DNA repair system consisting of 8-oxoguanine-DNA glycosylase (Ogg1), adenine-DNA glycosylase, and 8-oxo-dGTPase. We have cloned, overexpressed, and characterized mOgg1, the product of the murine ogg1 gene. mOgg1 is a DNA glycosylase/AP lyase belonging to the endonuclease III family of DNA repair enzymes. The AP lyase activity of mOgg1 is significantly lower than its glycosylase activity. mOgg1 releases 8-oxoG from DNA when paired with C, T, or G, but efficient DNA strand nicking is observed only with 8-oxoG:C. Binding of mOgg1 to oligonucleotides containing 8-oxoG:C is strong (K(D) = 51.5 nm), unlike other mispairs. The average residence time for mOgg1 bound to substrate containing 8-oxoG:C is 18.3 min; the time course for accumulation of the NaBH(4)-sensitive intermediate suggests a two-step reaction mechanism. Various analogs of 8-oxoG were tested as substrates for mOgg1. An electron-withdrawing or hydrogen bond acceptor moiety at C8 is required for efficient binding of mOgg1. A substituent at C6 and a keto group at C8 are required for cleavage. The proposed mechanism of 8-oxoG excision involves protonation of O(8) or the deoxyribose oxygen moiety.
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794, USA. dmitry@pharm.sunysb.edu
authors.forename: D O
authors.initials: DO
authors.lastname: Zharkov
authors.forename: T A
authors.initials: TA
authors.lastname: Rosenquist
authors.forename: S E
authors.initials: SE
authors.lastname: Gerchman
authors.forename: A P
authors.initials: AP
authors.lastname: Grollman
mesh_terms: D000818:Animals; D002384:Catalysis; D043382:DNA-Formamidopyrimidine Glycosylase; D007700:Kinetics; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase