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J. Biol. Chem., Vol. 279, Issue 18, 19217-19229, April 30, 2004

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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^*

Jeong-Yun Choi and F. Peter Guengerich

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

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, N²-isobutylG, N²-benzylG, and N²-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7^- showed a decrease of 10³ in k_cat/K_m 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-steady-state kinetic bursts with RT and T7^-, and N²-EtG eliminated the bursts. Large elemental effects with thio-dCTP(S) were observed with N²-EtG (6- and 72-fold decreases) but were much less with N²-MeG, indicating that the N²-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.

Received for publication, December 16, 2003 , and in revised form, February 22, 2004.

^* This work was supported in part by United States Public Health Service Grants R01 ES10375 and P30 ES00267 (to F. P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains MALDI-TOF mass spectra and capillary gel electrophoretograms of synthetic oligonucleotides.

To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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