THE OGG1 PROTEIN OF SACCHAROMYCES-CEREVISIAE - A 7,8-DIHYDRO-8-OXOGUANINE DNA GLYCOSYLASE AP LYASE WHOSE LYSINE-241 IS A CRITICAL RESIDUE FOR CATALYTIC ACTIVITY

The OGG1 gene of Saccharomyces cerevisiae codes dor a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and ,6-diamino-4-hydrox y-5-N-methylformamidopyrimidine (Fapy) from damaged DNA. In this paper , we have analysed the substrate specificity and the catalytic mechani sm of the Ogg1 protein acting on DNA subtrates containing 8-OxoG resid ues or apurinic/apyrimidinic (AP) sites. The Ogg1 protein displays a m arked preference for DNA duplexes containing 8-OxoG placed opposite a cytosine, the rank order for excision of 8-OxoG and cleavage efficienc ies being 8-OxoG/C > 8-OxoG/T >> 8-OxoG/G and 8-OxoG/A. The cleavage o f the DNA strand implies the excision of 8-OxoG followed by a beta-eli mination reaction at the 3'-side of the resulting AP site. The Ogg1 pr otein efficiently cleaves a DNA duplex where a preformed AP site is pl aced opposite a cytosine (AP/C). In contrast, AP/T, AP/A or AP/G subst rates are incised with a very low efficiency. Furthermore, cleavage of 8-OxoG/C or AP/C substrates implies the formation of a reaction inter mediate that is converted into a stable covalent adduct in the presenc e of sodium borohydre (NaBH4). Therefore, the Ogg1 protein is a eukary otic DNA glycosylase/AP lyase. Sequence homology searches reveal that Ogg1 probably shares a common ancestor gene with the endonuclease III of Escherichia coli. A consensus sequence indicates a highly conserved lysine residue, K120 of endonuclease III or K241 of Ogg1, respectivel y. Mutations of K241 to Gin (K241Q) and Arg (K241R) have been obtained after site directed mutagenesis of OGG1. Mutation K241Q completely ab olishes DNA glycosylase activity and covalent complex formation in the presence of NaBH4. However, the K241Q mutant still binds DNA duplexes containing 8-OxoG/C. In contrast, K241R mutation results in a catalyt ically active form of Ogg1. These results strongly suggest that the fr ee amino group of Lys241 is involved in the catalytic mechanism of the Ogg1 protein.