Catalytic mechanism and DNA substrate recognition of Escherichia coli MutY protein

被引:45
作者
Lu, AL
Yuen, DS
Cillo, J
机构
[1] Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore
关键词
D O I
10.1074/jbc.271.39.24138
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Escherichia coli MutY protein cleaves A/G- or A/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities, In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO containing DNA were cleaved by MutY with specificity constants (k(cat)/K-m) of 10 and 3.3 min(-1) mu M(-1), respectively, MutY(G116D) cleaved these DNAs 2,300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO containing DNA, but with lower efficiencies than the wild type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.
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收藏
页码:24138 / 24143
页数:6
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