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J. Biol. Chem., Vol. 281, Issue 18, 12315-12324, May 5, 2006

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Kinetic Evidence for Inefficient and Error-prone Bypass across Bulky N²-Guanine DNA Adducts by Human DNA Polymerase ^*

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

DNA polymerase (pol) has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol . Pol effectively bypassed N²-methyl (Me)G and N²-ethyl(Et)G, partially bypassed N²-isobutyl(Ib)G and N²-benzylG, and was blocked at N²-CH₂(2-naphthyl)G (N²-NaphG), N²-CH₂(9-anthracenyl)G (N²-AnthG), and N²-CH₂(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of k_cat/K_m for dCTP insertion opposite N²-G adducts according to size, with a maximal decrease opposite N²-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3–11-fold opposite adducts (highest with N²-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N²-IbG, N²-NaphG, and N²-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S_p-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N²-EtG and N²-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N²,N²-diMeG compared with N²-EtG by pol but not by pol is consistent with Hoogsteen base pairing by pol . Thus, polymerization by pol is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol may play a limited role in translesion synthesis on bulky N²-G adducts in cells.

Received for publication, January 5, 2006 , and in revised form, February 6, 2006.

^* 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 a MALDI-TOF mass spectrum and capillary gel electrophoretogram of the synthetic oligonucleotide containing N²,N²-diMeG and SDS-polyacrylamide gel electrophoretic analysis of purified DNA polymerase .

¹ Present address: Dept. of Pharmacology, College of Medicine, Ewha Womans University, 911-1 Mok-6-Dong, Yangcheon-Gu, Seoul, 158-710, Republic of Korea.

² 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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