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Originally published In Press as doi:10.1074/jbc.M106045200 on July 26, 2001

J. Biol. Chem., Vol. 276, Issue 41, 38090-38096, October 12, 2001
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Fidelity of Nucleotide Incorporation by Human Mitochondrial DNA Polymerase*

Allison A. Johnson and Kenneth A. JohnsonDagger

From the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712

We have examined the fidelity of polymerization catalyzed by the human mitochondrial DNA polymerase using wild-type and exonuclease-deficient (E200A mutation) forms of recombinant, reconstituted holoenzyme. Each of the four nucleotides bind and incorporate with similar kinetics; the average dissociation constant for ground state binding is 0.8 µM, and the average rate of polymerization is 37 s-1, defining a specificity constant kcat/Km = 4.6 × 107 M-1 s-1. Mismatched nucleotides show weaker ground-state nucleotide binding affinities ranging from 57 to 364 µM and slower rates of polymerization ranging from 0.013 to 1.16 s-1. The kinetic parameters yield fidelity estimates of 1 error out of 260,000 nucleotides for a T:T mismatch, 3563 for G:T, and 570,000 for C:T. The accessory subunit increases fidelity 14-fold by facilitating both ground-state binding and the incorporation rate of the correct A:T base pair compared with a T:T mismatch. Correctly base-paired DNA dissociates from the polymerase at a rate of 0.02 s-1 promoting processive polymerization. Thus, the mitochondrial DNA polymerase catalyzed incorporation with an average processivity of 1850, defined by the ratio of polymerization rate to the dissociation rate (37/0.02) and with an average fidelity of one error in 280,000 base pairs.

* This work was supported by National Institutes of Health Grant GM44613 (to K. A. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Inst. for Cellular and Molecular Biology, MBB 3.122, A4800, University of Texas, Austin, TX 78712. Tel.: 512-471-0434; Fax: 512-471-0435; E-mail: kajohnson@ mail.utexas.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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