Repair of O6-methylguanine and O4-methylthymine by the human and rat O6-methylguanine-DNA methyltransferases.

In order to compare the ability of the human and rat O6-methylguanine-DNA methyltransferases (transferases) to repair in vitro O6-methylguanine (O6-MeGua) and O4-methylthymine (O4-MeThy) residues, which are two mutagenic DNA adducts formed by alkylating agents, we have purified both proteins to homogeneity. Gel electrophoresis of the proteins shows that the O4-MeThy repair is due to the transfer of the methyl group from the alkylated base to the transferase molecules. However, both proteins repair with different efficiencies the O6-MeGua and O4-MeThy residues present in alkylated DNA, poly[d(G.C)], poly(dG.dC), or in alkylated poly[d(A.T)] and poly(dA.dT), respectively. Reaction of both proteins with either methylated residues follows a second-order kinetics. The rate constants are 1 x 10(9) M-1 min-1 for both proteins acting on O6-MeGua and 4.8 x 10(6) or 1.8 x 10(5) M-1 min-1 for the rat or human protein acting on O4-MeThy, respectively. The activity of the mammalian transferases on O4-MeThy present in a poly(dA.dT) substrate is inhibited by double-stranded DNA.