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J. Biol. Chem., Vol. 283, Issue 22, 15339-15348, May 30, 2008

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Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase^*

Jason D. Fowler¹, Jessica A. Brown², Kenneth A. Johnson^¶, and Zucai Suo^||**3

From the Department of Biochemistry, the Ohio State Biochemistry Program, the ^||Ohio State Biophysics Program, the ^**Molecular, Cellular and Developmental Biology Program, the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210 and the ^¶Institute for Cell and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712

Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase . Here we employed presteady state kinetic methods to directly investigate the incorporation of the 5'-triphosphorylated form of gemcitabine (dFdCTP), the excision of the incorporated monophosphorylated form (dFdCMP), and the bypass of template base dFdC catalyzed by human DNA polymerase . Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next 2 correct nucleotides by 214- and 7-fold, respectively. Moreover, the primer 3'-dFdCMP was excised with a 50-fold slower rate than the matched 3'-dCMP. When dFdC was encountered as a template base, DNA polymerase paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by 2 orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.

Received for publication, January 11, 2008 , and in revised form, March 10, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM079403 (to Z. S.) and GM044613 (to K. A. J.). 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. 1-3.

¹ Supported by American Heart Association Predoctoral Fellowship Grant GRT00004622.

² Supported by National Institutes of Health Chemistry and Biology Interface Program at The Ohio State University (Grant 5 T32 GM008512-11).

³ 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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