A spontaneous mutator strain ofEscherichia coli (fpg mutY) was used to clone the OGG] gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7, (fpg) harboring pYSB110 was used to purify the Oggl protein of S. cerevisiae to apparent homogeneity. The Oggl protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. TheOggl protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Oggl protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Oggl protein is probably due to the excision of 8-OxoG followed by a a-elimination at the resulting apurinic/apyrimidinic site.Reactive oxygen species formed in cells as by-products of aerobic metabolism or during oxidative stress have been suggested to play a role in biological processes such as mutagenesis, carcinogenesis, and aging (1, 2). Hydroxyl radical (OH') and singlet oxygen (102) are highly reactive and produce complex patterns of DNA modifications (3, 4). Several lines of evidence suggest that an oxidatively damaged form of guanine,7,, is highly mutagenic in vitro and in vivo (5, 6). Escherichia coli possesses two DNA glycosylase activities that prevent spontaneous mutagenesis by 8-OxoG: the Fpg protein, which excises 8-OxoG residues in DNA (4, 7-9), and the MutY protein, which excises the adenine residues incorporated opposite 8-OxoG (7, 10, 11). The Fpg protein also releases imidazole ring-opened purines such as 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Fapy) (9). Inactivation of both the fg (mutM) and the mutY (micA) genes of E. coli results in a strong GC --TA spontaneous mutator phenotype (7,12,13

The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamin0-4-hydroxy-SNmethylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites. Furthermore, the expression of the fpg4 gene in fpg and mutY mutants of E. coli suppresses their spontaneous GC + TA mutator phenotype.The similarity of the activity of the two Fpg proteins and its conservation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.