Catalytic Mechanism and DNA Substrate Recognition of Escherichia coli MutY Protein

A-Lien Lu,David S Yuen,Jason Cillo

doi:10.1074/jbc.271.39.24138

A-Lien Lu, David S Yuen + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.39.24138

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Abstract

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 (kcat/Km) of 10 and 3.3 min-1 microM-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.

Highlights

Oxidative stress and metabolic processes that produce reactive oxygen species have been implicated in aging and a number of diseases including cancer [1, 2]
In Escherichia coli, the proteins MutY, MutM, and MutT are involved in defending against the mutagenic effects of GO lesions [4, 5]
The combined glycosylase/AP lyase activity of mutant MutY(G116D) with A/G-containing DNA was more severely affected than it was with A/GO-containing DNA. These results suggest that this conserved region is in the proximity of the substrate binding and/or catalytic sites

Summary

EXPERIMENTAL PROCEDURES

Bacteria—E. coli PR70 (SuϪ lacZ X74 galU galK Smr micA68::Tn10Kan) and GBE791 [lacIp4000 (LacIq) lacZp4008 (Lac L8) srlC-300::Tn10 ␭Ϫ IN (rrnD-rrnE)1] were obtained from M. Mutant MutY(G116D) and MutY(G116A) were purified from about 40 g of E. coli GBE943 cells harboring overproduction plasmids pMGD116 and pMGA116, respectively, in a similar manner as with wild-type enzyme. The standard reaction mixture contained 20 mM Tris-HCl (pH 7.6), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 2.9% glycerol, 20 ng of poly(dI-dC), and 1.8 fmol of labeled 20-base pair oligonucleotide in a total volume of 20 ␮l. The apparent dissociation constants (Kd values) of mutant MutY and DNA containing A/GO or A/G mismatches were determined using a range of protein concentrations as described previously [17]. 3Ј-end-labeled 20-base pair oligonucleotides (1.8 fmol) were incubated with various concentrations of MutY protein as described in the binding assay, except that poly(dI-dC) was omitted from the reaction in a total volume of 10 ␮l. After incubation at 37 °C for 30 min, the products were separated on a 12 or 15% polyacrylamide gel in the presence of SDS (SDS-polyacrylamide gel electrophoresis) according to Laemmli [45], and the gel was dried and autoradiographed

RESULTS

DISCUSSION

21 Ϯ 2 17 Ϯ 2 100 Ϯ 10 120 Ϯ 10