Substrate specificity of Escherichia coli MutY protein.

The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme. In contrast to a recent report [Lu, A.-L., et al. (1995) J. Biol. Chem. 270, 23582], dA:8-oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (kcat/Km) for dA:8-oxo-dG and dA:dG of 39 600 x 10(-6) and 383 x 10(-6) (min-1 nM-1), respectively. kcat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0. The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested. Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 x 10(-6) (min-1 nM-1), whereas replacement of dA by 2'-O-methyladenosine virtually abolished enzymatic activity. Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2'-O-methylguanosine generated a substrate with a low specificity constant. Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate. Duplexes containing a carbocyclic analog of dA were not cleaved. A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme.