Catalytic Mechanism and DNA Substrate Recognition of Escherichia coli MutY Protein

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.

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

“…Covalent trapping of mtMYH with A/8-oxoG in the presence of sodium borohydride was performed using the method of Lu et al (51). Protein samples were incubated with 1.8 fmol 3′-end labeled 20mer duplex DNA in a 20 µl reaction mixture containing the same buffer used in the glycosylase assay and different concentrations of NaBH 4 .…”

Reaction Intermediates in the Catalytic Mechanism of Escherichia coli MutY DNA Glycosylase

“…MutY (400 nM) was incubated with 2 nM substrate DNA containing a G:8-oxoG mismatch. The reaction mixture was incubated at 37°C, and aliquots were withdrawn at specific time points (1,5,15,30,60, and 300 min, lanes 2-7, respectively). Piperidine was added to these aliquots, and incubation continued at 85°C for 15 min.…”

“…Although there is a consensus on the glycosylase base excision activity of MutY, the AP lyase activity of MutY has remained poorly defined. Biochemical evidence has been presented that argues for MutY being a monofunctional glycosylase (19 -21), whereas other data are consistent with the enzyme being a glycosylase with a concomitant AP lyase activity (30,31).…”

Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans

“…The possession of AP lyase activity by MutY is controversial. MutY AP lyase activity has been detected by some researchers (15,22,25,26,29,30,48) but not by other groups (1,7,31,34,44). Although MutY lacks the conserved lysine, there is evidence that MutY may be a bifunctional glycosylase.…”

“…MutY can cleave DNA containing an unmodified AP site (30,54). The enzyme also forms a covalent complex with its DNA substrates in the presence of sodium borohydride (15,26,30,51,57), a diagnostic tool for bifunctional glycosylase/AP lyase (13,40,44). Lys142 of MutY Ec has been shown to form the Schiff base intermediate with DNA (52,54,57); however, K142A mutant MutY has glycosylase activity (52,54) and can promote ␤/␦ elimination with AP-containing DNA (54).…”