FUNCTIONAL MAPPING OF THE ECORV DNA METHYLTRANSFERASE BY RANDOM MUTAGENESIS AND SCREENING FOR CATALYTICALLY INACTIVE MUTANTS

M.EcoRV is an alpha-adenine DNA methyltransferase. According to struct ure predictions, the enzyme consists of a catalytic domain, which has a structure similar to all other DNA-methyltransferases, and a smaller DNA-recognition domain. We have investigated this enzyme by random mu tagenesis, using error-prone FOR, followed by selection for catalytica lly inactive mutants. 20 single mutants were identified that are compl etely inactive in vivo as His(6)- and GST-fusion proteins. 13 of them could be overexpressed and purified. All of these mutants are also ina ctive in vitro. 5 of the mutations are located near the putative bindi ng site for a flipped adenine residue (C192R, D193G, E212G, W231R, N23 9H), All of these variants bind to DNA, demonstrating the importance o f this region of the protein in catalysis. Only the W231R mutant could be purified with high yields. It binds to DNA and AdoMet and, thus, b ehaves like a bona fide active site mutant. According to the structure prediction Trp231 corresponds to Val121 in M.Hhal, which forms a hydr ophobic contact to the flipped target cytosine. 4 of the remaining pur ified variants are located within a small region of the putative DNA-r ecognition domain (F115S, F117L, S121P, C122Y). F117L, S121P and C122Y are unable to bind to DNA, suggesting a critical role of this region in DNA binding. Taken together, these results are in good agreement wi th the structural model of M.EcoRV.