Role for lysine 142 in the excision of adenine from A : G mispairs by MutYDNA glycosylase of Escherichia coli

MutY participates in the repair of oxidatively damaged DNA by excising aden ine front 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 mu tant protein in which Lys-142 is replaced by glutamine, Using the rifampici n resistance assay, MutY K142Q was shown to complement the mutY mutator phe notype to the same extent as wild-type MutY. Although MutY K142Q does not f orm 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, meas ured in single turnover experiments, was not significantly affected. At pH < 6.0, the activity of MutY K142Q on the dA:dG mispair was approximately th e same as for wild-type protein, suggesting that a dc(anti) to dG(syn) tran sition is effected at low pH. The three-dimensional structure of the cataly tic domain of MutY K142Q, determined at 1.35 <Angstrom> resolution, shows n o significant differences between wild-type and mutant protein, indicating that Lys-142 is not critical for maintaining the conformation of MutY. We c onclude that Lys-142 recognizes guanine in the dA:dG mispair, helping posit ion this residue in the syn conformation and facilitating binding of substr ate DNA, Lys-142 is not involved in the catalytic steps of base excision.