REQUIREMENT OF THE PRO-CYS-HIS-ARG SEQUENCE FOR O-6 METHYLGUANINE-DNAMETHYLTRANSFERASE ACTIVITY REVEALED BY SATURATION MUTAGENESIS WITH NEGATIVE AND POSITIVE SCREENING

O-6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O-6-methylguanine and O-4-methylthymine of DNA to a cystei ne residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conse rved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the sign ificance of the conserved sequence, amino acid substitutions were intr oduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant f orms of the enzyme were examined. When cysteine-139, to which methyl t ransfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-pos itive revertants, isolated from one of the negative mutants, had resto red codons for cysteine. Thus the cysteine residue is essential for ac ceptance of the methyl group and is not replaceable by other amino aci ds. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidi ne-140 and arginine-141 sites, all the positive revertants isolated ca rried codons for amino acids identical to those of the wild-type prote in. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutan t forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the prote in. With valine-142 substitutions there were seven types of positive r evertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activi ty. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.