Catalytic and DNA binding properties of the Ogg1 protein of Saccharomyces cerevisiae: Comparison between the wild type and the K241R and K241Q active-site mutant proteins

The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA gly cosylases and apurinic/apyrimidinic site (AP) lyases, the signature of whic h is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K m otif spans residues 225-260 and the conserved lysine is K241. In this study , we have purified the K241R and K241Q mutant proteins and compared their c atalytic and DNA binding properties to that of the wild-type yOgg1. The res ults show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8- OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-3) 5 x 10(-3) min(-1 ) nM(-1) for the wild-type and the K241R protein, respectively. On the othe r hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activ ities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wi ld-type DNA binding properties. K-dapp values for binding of [8-OxG.C; C] a re 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respe ctively. The results also show that AP site analogues such as 1,3-propanedi ol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wi ld-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cyt osine placed opposite Pr was strongly protected. The same footprints were o bserved with the K241R and K241Q proteins, confirming their wild-type DNA b inding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizab le substrates such as 8-OxoG or an AP site.