SUBSTRATE-SPECIFICITY OF ESCHERICHIA-COLI MUTY PROTEIN

The MutY protein of Escherichia coil removes mismatched deoxyadenine r esidues from DNA. In this study, duplex oligodeoxynucleotides containi ng modified bases are used as model substrates for this enzyme. In con trast 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 (k(cat)/K-m) for dA:8 -oxo-dG and dA:dG of 39 600 x 10(-6) and 383 x 10(-6) (min(-1) nM(-1)) , respectively. k(cat) for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG w as unaffected over a pH range of 5.5-8.0. The presence of an 8-oxo fun ction in dG increased significantly the rate of removal of dA from all substrates tested. Replacement of dA by rA reduced the specificity co nstant of dA:8-oxo-dG to 294 x 10(-6) (min(-1) nM(-1)), whereas replac ement of dA by 2'-O-methyladenosine virtually abolished enzymatic acti vity. Modifications of the dG moiety generally were better tolerated t han those of dA; however, introduction of a methyl ether at the 6 posi tion of dG produced a noncleavable substrate and replacement of dG by 2'-O-methylguanosine generated a substrate with a low specificity cons tant. Rates of cleavage of duplexes containing dA:dC and dA:tetrahydro furan were three orders of magnitude lower than the reference substrat e. 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.