Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase

Genomic DNA is prone to oxidation by reactive oxygen species. A major produ ct of DNA oxidation is the miscoding base 8-oxoguanine (8-oxoG). The mutage nic effects of 8-oxoG in mammalian cells are prevented by a DNA repair syst em consisting of 8-oxoguanine-DNA glycosylase (Ogg1), adenine-DNA glycosyla se, and 8-oxo-dGTPase. We have cloned, overexpressed, and characterized mOg g1, the product of the murine ogg1 gene. mOgg1 is a DNA glycosylase/AP lyas e belonging to the endonuclease III family of DNA repair enzymes. The AP ly ase 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 olig onucleotides containing 8-oxoG:C is strong (K-D = 51.5 nM), unlike other mi spairs. The average residence time for mOgg1 bound to substrate containing 8-oxoG:C is 18.3 min; the time course for accumulation of the NaBH4-sensiti ve intermediate suggests a two-step reaction mechanism. Various analogs of 8-oxoG were tested as substrates for mOgg1. An electron-withdrawing or hydr ogen bond acceptor moiety at CS 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 de oxyribose oxygen moiety.