Studies of Escherichia coli micA (mutY)-dependent mismatch repair

Abstract

Escherichia coli possesses two repair systems to correct A/G mismatches. One is directed by dam methylation, the other is dependent on micA (mutY) (Modrich, 1991). The micA (mutY)-dependent repair pathway is specific for A/G and A/C mismatches with 20-fold higher activity on A/G mismatches. It requires DNA polymerase I for the repair synthesis and the repair tract does not exceed twelve nucleotides. It is not dependent on mutHLS, uvrE, recBC, recF, recJ, recN, mutT, or dnaE gene functions. micA gene was cloned and sequenced. Two open reading frames (ORFs) were found in a 2.3-kb EcoRI fragment. One ORF codes for a 39.1-kD protein which is the micA gene as shown by deletion analysis. The nucleotide sequence of micA gene shows that it is the same gene as mutY. The second ORF is located upstream of the micA gene with opposite orientation and encodes a 25.7-kD protein. The function of this protein is unknown. The micA (mutY) gene was cloned into an expression vector pKK223-3 and its product was purified from this overproducing strain. After four chromatographic steps, MicA (MutY) protein was purified 18 fold to {dollar}>{dollar}99% homogeneity. Purified MicA (MutY) protein has both N-glycosylase activity, which removes the adenines from the mismatches, and apurinic/apyrimidinic(AP)endonuclease activity, which cleaves the first phosphodiester bond 3{dollar}\sp\prime{dollar} to the apurinic site. The protein with the active nicking activity is a monomer as shown by gel filtration chromatography. Furthermore, in the presence of iron and sulfide, both glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from a sodium dodecyl sulfate (SDS)-polyacrylamide gel. These findings suggest that the MicA (MutY) protein, like E. coli endonuclease III, is an iron-sulfur protein. The potential DNA contacts of MicA (MutY) on the A/G containing DNA were studied by DNase I footprinting, methylation interference and ethylation interference. The protein covers about 12 bp of the A/G-containing DNA and has more contacts on the dG-strand. The mechanism of the micA (mutY)-dependent repair pathway is discussed.