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 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 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].