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J. Biol. Chem., Vol. 281, Issue 30, 21062-21072, July 28, 2006

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Translesion Synthesis across Bulky N²-Alkyl Guanine DNA Adducts by Human DNA Polymerase ^*

Jeong-Yun Choi, Karen C. Angel, and F. Peter Guengerich¹

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and the Department of Pharmacology, College of Medicine, Ewha Womans University, 911-1 Mok-6-Dong, Yangcheon-Gu, Seoul 158-710, Republic of Korea

DNA polymerase (pol) is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol , and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol was studied with a series of eight N²-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH₂(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k_cat/K_m) were similar for insertion of dCTP opposite the lesions and for extension beyond the N²-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k_cat/K_m values 10^-3 less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (k_p 5.9-10.3 s^-1). The rapid bursts were abolished opposite BPG when -thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols and ; pol is the most resistant to N²-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N²-adducts, particularly the largest (N²-BPG).

Received for publication, March 9, 2006 , and in revised form, April 6, 2006.

^* This work was supported by United States Public Health Services 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 SDS-polyacrylamide gel electrophoretic analysis of purified DNA polymerase (supplemental Figs. S1 and S2).

¹ 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 Avenues, Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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