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J. Biol. Chem., Vol. 282, Issue 11, 8188-8198, March 16, 2007

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Mechanism of Abasic Lesion Bypass Catalyzed by a Y-family DNA Polymerase^*

Kevin A. Fiala¹, Cameron D. Hypes, and Zucai Suo^¶||2

From the Department of Biochemistry, the Ohio State Biochemistry Program, the ^¶Molecular, Cellular and Developmental Biology Program, and the ^||Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210

The 3 million-base pair genome of Sulfolobus solfataricus likely undergoes depurination/depyrimidination frequently in vivo. These unrepaired abasic lesions are expected to be bypassed by Dpo4, the only Y-family DNA polymerase from S. solfataricus. Interestingly, these error-prone Y-family enzymes have been shown to be physiologically vital in reducing the potentially negative consequences of DNA damage while paradoxically promoting carcinogenesis. Here we used Dpo4 as a model Y-family polymerase to establish the mechanistic basis for DNA lesion bypass. While showing efficient bypass, Dpo4 paused when incorporating nucleotides directly opposite and one position downstream from an abasic lesion because of a drop of several orders of magnitude in catalytic efficiency. Moreover, in disagreement with a previous structural report, Dpo4-catalyzed abasic bypass involves robust competition between the A-rule and the lesion loop-out mechanism and is governed by the local DNA sequence. Analysis of the strong pause sites revealed biphasic kinetics for incorporation indicating that Dpo4 primarily formed a nonproductive complex with DNA that converted slowly to a productive complex. These strong pause sites are mutational hot spots with the embedded lesion even affecting the efficiency of five to six downstream incorporations. Our results suggest that abasic lesion bypass requires tight regulation to maintain genomic stability.

Received for publication, November 20, 2006 , and in revised form, January 3, 2007.

^* This work was supported in part by the National Science Foundation Career Award MCB-0447899 (to Z. S.). 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. 1 and 2 and Tables I–IV.

¹ Recipient of American Heart Association Predoctoral Fellowship 0415129B and the Herta Camerer Gross Graduate Research Fellowship.

² To whom correspondence should be addressed: 740 Biological Sciences, 484 West 12th Ave., Columbus, OH 43210. Tel.: 614-688-3706; Fax: 614-292-6773; E-mail: suo.3{at}osu.edu.

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