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J. Biol. Chem., Vol. 281, Issue 47, 36241-36248, November 24, 2006

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Incorporation and Replication of 8-Oxo-deoxyguanosine by the Human Mitochondrial DNA Polymerase^*

From the Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 µM^-1 s^-1, a value 10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 µM^-1 s^-1, a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.

Received for publication, August 21, 2006 , and in revised form, September 27, 2006.

^* This work was supported by National Institutes of Health Grant R01 GM044613 and the Welch Foundation Grant F-1604. 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.

¹ To whom correspondence should be addressed: Institute for Cellular and Molecular Biology, University of Texas at Austin, 2500 Speedway, A4800, Austin, TX 78735. Tel.: 512-471-0434; Fax: 512-471-0435; E-mail: kajohnson{at}mail.utexas.edu.

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