Role for Lysine 142 in the Excision of Adenine from A:G Mispairs by MutY DNA Glycosylase of Escherichia coli

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. We have investigated the properties of a mutant protein in which Lys-142 is replaced by glutamine. Using the rifampicin resistance assay, MutY K142Q was shown to complement the mutY mutator phenotype to the same extent as wild-type MutY. Although MutY K142Q does not form a Schiff base with DNA, it retains in part the catalytic properties of wild-type enzyme. The K142Q mutation selectively impairs processing of DNA containing dA:dG mispairs but not that of substrates containing dA:8-oxodG. Decreased substrate processing is mediated primarily via an increase in K D (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-type protein, suggesting that a dG(anti) to dG(syn) transition is effected at low pH. The three-dimensional structure of the catalytic domain of MutY K142Q, determined at 1.35 Å resolution, shows no significant differences between wild-type and mutant protein, indicating that Lys-142 is not critical for maintaining the conformation of MutY. We conclude that Lys-142 recognizes guanine in the dA:dG mispair, helping position this residue in the syn conformation and facilitating binding of substrate DNA. Lys-142 is not involved in the catalytic steps of base excision.