文摘
MutY participates in the repair of oxidatively damaged DNA by excising adenine from dA:dG and dA:8-oxodG mispairs; this DNA glycosylase can be cross-linked to DNA through Lys-142. Wehave investigated the properties of a mutant protein in which Lys-142 is replaced by glutamine. Using therifampicin resistance assay, MutY K142Q was shown to complement the mutY mutator phenotype to thesame extent as wild-type MutY. Although MutY K142Q does not form a Schiff base with DNA, it retainsin part the catalytic properties of wild-type enzyme. The K142Q mutation selectively impairs processingof DNA containing dA:dG mispairs but not that of substrates containing dA:8-oxodG. Decreased substrateprocessing is mediated primarily via an increase in KD (21.8 nM for MutY vs 298 nM for MutY K142Q).The catalytic constant, measured in single turnover experiments, was not significantly affected. At pH <6.0, the activity of MutY K142Q on the dA:dG mispair was approximately the same as for wild-typeprotein, suggesting that a dG(anti) to dG(syn) transition is effected at low pH. The three-dimensionalstructure of the catalytic domain of MutY K142Q, determined at 1.35 Å resolution, shows no significantdifferences between wild-type and mutant protein, indicating that Lys-142 is not critical for maintainingthe conformation of MutY. We conclude that Lys-142 recognizes guanine in the dA:dG mispair, helpingposition this residue in the syn conformation and facilitating binding of substrate DNA. Lys-142 is notinvolved in the catalytic steps of base excision.