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

• Published in: The Journal of biological chemistry Vol. 279; no. 18; pp. 19217 - 19229
• Main Authors: Choi, Jeong-Yun, Guengerich, F Peter
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30.04.2004
• Subjects: Alkylation; Bacteriophage T7 - enzymology; Bacteriophage T7 - genetics; Base Pair Mismatch; Catalysis; DNA Adducts - chemistry; DNA Adducts - pharmacology; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; Guanine - analogs & derivatives; Guanine - chemistry; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; HIV-1 - enzymology; HIV-1 - genetics; Kinetics; Mutation; Substrate Specificity; Viral Proteins - metabolism; Virus Replication - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M313759200

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 N 2 -methyl(Me)G and readily extended primers but were strongly blocked by N 2 -ethyl(Et)G, N 2 -isobutylG, N 2 -benzylG, and N 2 -methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7 - showed a decrease of 10 3 in k cat / K m for dCTP incorporation opposite N 2 -MeG and a further large decrease opposite N 2 -EtG. Misincorporation frequency was increased 10 2 -10 3 -fold by a Me group and another ∼10 3 -fold by an Et group. dATP was preferentially incorporated opposite bulky N 2 -alkylG molecules. N 2 -MeG attenuated the pre-steady-state kinetic bursts with RT and T7 - , and N 2 -EtG eliminated the bursts. Large elemental effects with thio-dCTP(αS) were observed with N 2 -EtG (6- and 72-fold decreases) but were much less with N 2 -MeG, indicating that the N 2 -Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K d (dCTP) and K d (DNA) and k off 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.