ESCHERICHIA-COLI MUTY PROTEIN HAS BOTH N-GLYCOSYLASE AND APURINIC APYRIMIDINIC ENDONUCLEASE ACTIVITIES ON A-CIRCLE-C AND A-CIRCLE-G MISPAIRS

In Escherichia coli the mutY (or mic.A)-dependent DNA mismatch repair pathway can convert A.G and A.C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of A.G and A.C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein. DNA fragments with A.C mismatches were 20-fold less active than DNA with A.G mispairs as a substrate for purified MutY.