Specificity of DNA repair methyltransferases determined by competitive inactivation with oligonucleotide substrates: evidence that Escherichia coli Ada repairs O6-methylguanine and O4-methylthymine with similar efficiency.

DNA repair methyltransferases (MTases) are stoichiometric acceptor molecules that are irreversibly inactivated in the course of removing a methyl group from O6-methylguanine (meG)-DNA or O4-methylthymine (meT)-DNA. A new assay has been developed to determine the relative efficiency of repair of meG and meT. The assay is based on the deprotection of methylated restriction sites in synthetic oligonucleotides and can be used to measure meG repair or meT repair directly. More importantly, relative repair efficiencies can be measured in competition experiments, using each of the methylated oligomers in turn as an inhibitor of repair for the other. Relative repair rates are determined by numerical solution of the coupled rate equations that describe this competition to the experimental data. We find that the human MTase repairs meT about 35-fold less well than meG, qualitatively similar to earlier studies. Contrary to previous reports, however, we find that Escherichia coli Ada repairs meG and meT with nearly equal efficiency. This finding, in conjunction with other recent reports, may indicate that low meT repair is a relatively unusual characteristic of the human homolog.

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.