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J. Biol. Chem., Vol. 282, Issue 2, 1397-1408, January 12, 2007

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Increased Flexibility Enhances Misincorporation

TEMPERATURE EFFECTS ON NUCLEOTIDE INCORPORATION OPPOSITE A BULKY CARCINOGEN-DNA ADDUCT BY A Y-FAMILY DNA POLYMERASE^*

Rebecca A. Perlow-Poehnelt¹, Ilya Likhterov², Lihua Wang, David A. Scicchitano, Nicholas E. Geacintov, and Suse Broyde³

From the Departments of Biology and Chemistry, New York University, New York, New York 10003

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N²-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 °C and results compared with an earlier study at 37 °C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 °C compared with 37 °C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 °C compared with 37 °C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the k_cat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

Received for publication, July 17, 2006 , and in revised form, September 28, 2006.

^* This work was supported by National Institutes of Health Grants CA28038 (to S. B.), ES10581 (to D. A. S.), and CA099194 (to N. E. 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 supplemental Figs. S1-S7 and Tables S1 and S2.

¹ Current address: Molecular Systems, Merck Research Laboratories, Merck & Co., Inc., P. O. Box 4, West Point, PA 19486.

² Current address: Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021.

³ To whom correspondence should be addressed: 100 Washington Square East, 1009 Silver Center, New York, NY 10003. Tel.: 212-998-8231; Fax: 212-995-4015; E-mail: broyde{at}nyu.edu.

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