Effect of the O6 substituent on misincorporation kinetics catalyzed by DNA polymerases at O6-methylguanine and O6-benzylguanine

Misincorporation at a DNA-carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O-6-alkylguanine. Replication of primer/templates containing guanine (G), O-6-methylguanine (O-6-MeG), or O-6-benzylguanine (O-6-BzG) was assessed using T7 DNA polymerase exo(-) (T7(-)) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7- and RT preferentially incorporated dTTP opposite O-6-MeG and O-6-BzG. The incorporation efficiencies (k(cat)/K-m) were less for O-6-BzG than O-6-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase. i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O-6-alkylG-containing substrates. Extension of the O-6-BzG-containing duplexes was much more difficult for both polymerases as compared to O-6-MeG, except when RT easily, extended the O-6-BzG:T base pair. The K-d(dNTP) for binding of dCTP or dTTP to a RT.DNA complex containing O-6-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA The K-d(dNTP) for a RT.DNA complex containing O-6-BzG was 50-fold greater. In conclusion, the bulkier O-6-BzG is a greater block to polymerization by T7(-) and RT than is O-6-MeG, but some polymerization does occur with an O-6-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O-6-BzG-containing DNA, unlike the case of O-6-MeG. These results and others regarding polymerase stalling opposite O-6-MeG and O-6-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039-1050].