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

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• 作者：Dmitry O. Zharkov ; Rotem Gilboa ; Ishay Yagil ; Jadwiga H. Kycia ; Sue Ellen Gerchman ; Gil Shoham ; and Arthur P. Grollman
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 5, 2000
• 年：2000
• 卷：39
• 期：48
• 页码：14768 - 14778
• 全文大小：271K
• 年卷期：v.39,no.48(December 5, 2000)
• ISSN：1520-4995

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 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-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.