Analysis of the Effect of Bulk at N2-Alkylguanine DNA Adducts on Catalytic Efficiency and Fidelity of the Processive DNA Polymerases Bacteriophage T7 Exonuclease- and HIV-1 Reverse Transcriptase

Abstract

The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease- (T7-). RT and T7 - effectively bypassed N2-methyl(Me)G and readily extended primers but were strongly blocked by N2-ethyl(Et)G, N 2-isobutylG, N2-benzylG, and N 2-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7- showed a decrease of 103 in kcat/Km for dCTP incorporation opposite N2-MeG and a further large decrease opposite N2-EtG. Misincorporation frequency was increased 102-103-fold by a Me group and another ∼103-fold by an Et group. dATP was preferentially incorporated opposite bulky N2-alkylG molecules. N2-MeG attenuated the pre-steadystate kinetic bursts with RT and T7-, and N2-EtG eliminated the bursts. Large elemental effects with thio-dCTP(αS) were observed with N2-EtG (6- and 72-fold decreases) but were much less with N2-MeG, indicating that the N 2-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of Kd(dCTP) and Kd(DNA) and koff rates of DNA substrates from RT and T7- indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.