Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase.

Abstract

The O6-methylguanine-DNA methyltransferase of Escherichia coli acts rapidly and stoichiometrically to convert a mutagenic O6-methylguanine residue in DNA to unsubstituted guanine. Even at low protein concentrations and in the absence of any cofactors, the transfer of a methyl group to one of the protein's own cysteine residues occurs in less than 2 s at 37 degrees C. The entire kinetic process can be followed experimentally at 5 degrees C. Formation of S-methylcysteine in the protein is accompanied by loss of activity and accounts for the exceptional suicide kinetics of this enzyme as well as for the sharp saturation of O6-methylguanine repair observed in vivo. The enzyme can remove greater than 98% of the methyl groups from O6-methylguanine present in alkylated DNA, but leaves N-alkylated purines untouched. Single-stranded DNA containing O6-methylguanine is a poor substrate, with the methyl transfer occurring at approximately 0.1% of the rate for duplex DNA. This latter observation may explain the high frequency of mutations induced by alkylating agents at DNA replication forks.