O6-alkylguanine-DNA alkyltransferases repair O6-methylguanine in DNA with Michaelis-Menten-like kinetics.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs O(6)-methylguanine (O(6)mG) by transferring the methyl group from the DNA to a cysteine residue on the protein. The kinetics of this reaction was examined by reacting an excess of AGT (0-300 nM) with [5'-(32)P]-labeled oligodeoxynucleotides (0.5 nM) of the sequence 5'-CGT GGC GCT YZA GGC GTG AGC-3' in which Y or Z was G or O(6)mG, annealed to its complementary strand. The reactions, conducted at 25 degrees C, were quenched by the addition of 0.1 N NaOH at various times, and the extents of reaction were monitored by ion exchange HPLC with radiochemical detection. The time courses followed first-order kinetics. The first-order rate constants were plotted against the initial concentration of AGT and fitted to the hyperbolic equation k(obs) = k(inact)[AGT](0)/(K(S) + [AGT](0)). The K(S) values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O(6)mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O(6)mG in DNA. The proteins reacted with DNA in which Y = O(6)mG and Z = G faster than Y = G and Z = O(6)mG due to an approximately 10-fold increase in k(inact). These results suggest that the sequence specificity in the repair of O(6)mG is manifested in the methyl transfer not in the O(6)mG recognition step.