O⁶-Alkylguanine-DNA Alkyltransferases Repair O ⁶-Methylguanine in DNA with Michaelis-Menten-like Kinetics

O⁶-Alkylguanine-DNA alkyltransferase (AGT) repairs O ⁶-methylguanine (O⁶mG) 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′-³²P]-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 O⁶mG, annealed to its complementary strand. The reactions, conducted at 25 °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] _o/(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 O⁶mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O⁶mG in DNA. The proteins reacted with DNA in which Y = O⁶mG and Z = G faster than Y = G and Z = O⁶mG due to an approximately 10-fold increase in k _inact. These results suggest that the sequence specificity in the repair of O⁶mG is manifested in the methyl transfer not in the O ⁶mG recognition step.