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.
Highlights
Genomic DNA is prone to oxidation by reactive oxygen species
DNA glycosylases have been classified as monofunctional or bifunctional (EndoIII, Fpg, bacteriophage T4 endonuclease V, and others) based on their ability to catalyze -elimination after base excision and to form borohydride-sensitive covalent intermediates [28]
Most bifunctional DNA glycosylases possess coupled glycosylase and AP lyase activities, catalyzing strand nicking at approximately the same rate as base release, and are readily cross-linked to their substrates by treatment with NaBH4
Summary
8-oxodG, 8-oxo-7,8-dihydro-2Јdeoxyguanosine; AP, apurinic/apyrimidinic; EndoIII, endonuclease III; Me-FaPy-G, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine; PAGE, polyacrylamide gel electrophoresis. Several DNA repair enzymes known as the “GO system” prevent mutagenesis via 8-oxoG [9]. This system consists of MutT, an 8-oxodGTPase that prevents incorporation of 8-oxodG into DNA from the triphosphate pool [10]; Fpg (MutM), an 8-oxoguanine-DNA glycosylase that preferentially excises 8-oxoG paired with C [11]; and MutY, an adenine-DNA glycosylase that preferentially excises A paired with 8-oxoG [9], initiating a round of base excision repair that restores the 8-oxoG:C pair, a substrate for Fpg. Eukaryotic homologs have been discovered for the components of the GO system; these include the 8-oxoguanine-DNA glycosylases, Ogg and Ogg, isolated from yeast [12,13,14]. We report marked differences in the efficiency of several steps in the mOgg1-catalyzed reaction with damaged DNA
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