<i>O</i>⁶-Alkylguanine−DNA Alkyltransferases Repair <i>O</i>⁶-Methylguanine in DNA with Michaelis−Menten-like Kinetics

Abstract

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O6-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 kobs = kinact[AGT]0/(KS + [AGT]0). The KS 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 kinact. These results suggest that the sequence specificity in the repair of O6mG is manifested in the methyl transfer not in the O6mG recognition step.