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J. Biol. Chem., Vol. 282, Issue 44, 31982-31989, November 2, 2007

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Fidelity and Processivity of Reverse Transcription by the Human Mitochondrial DNA Polymerase^*

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

We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol ) during reverse transcription, employing a synthetic oligonucleotide consisting of a DNA primer and an RNA template. In comparison with the kinetic parameters observed with a DNA template, the rate of correct deoxynucleotide incorporation was reduced 25-fold (5.5 ± 0.2 s^–1), whereas the dissociation constant (K_d) for nucleotide binding was increased 4-fold (12 ± 1 µM). In addition, discrimination against mismatches was reduced 20-fold to only 15,000 on average. The proofreading exonuclease favored the removal of an incorrect nucleotide (0.0021 ± 0.0002 s^–1 for correct versus 0.034 ± 0.004 s^–1 for incorrect), and the partitioning between incorporation beyond a mismatch (5.5 x 10 ^–5 ± 0.4 x 10 ^–5 s^–1), and exonuclease removal of that mismatch favors removal of the mismatch. These data suggest that the "reverse transcriptase activity" of mitochondrial polymerase could be physiologically relevant. However, the enzyme stalls and is unable to efficiently incorporate beyond a single nucleotide with an RNA template. Additionally, we present a refined method for calculating net discrimination, which more accurately describes the contributions of correct and incorrect incorporation. The biological and biotechnological significance of these results are discussed.

Received for publication, July 2, 2007 , and in revised form, August 16, 2007.

^* This work was supported by National Institutes of Health Grant GM 044613 and the Welch Foundation 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 President of KinTek Corp., the manufacturer of the rapid quench-flow instruments used in this study. To whom correspondence should be addressed. Dept. of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology, University of Texas, 2500 Speedway, Austin, TX 78712. Tel.: 512-471-0434; Fax: 512-471-0435; E-mail: kajohnson{at}mail.utexas.edu.

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