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J. Biol. Chem., Vol. 283, Issue 21, 14411-14416, May 23, 2008

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Base Pair Hydrogen Bonds Are Essential for Proofreading Selectivity by the Human Mitochondrial DNA Polymerase^*

Harold R Lee, Sandra A. Helquist, Eric T. Kool, and Kenneth A. Johnson¹

From the Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712 and Department of Chemistry, Stanford University, Stanford, California 94305

We have characterized the role of Watson-Crick hydrogen bonding in the 3'-terminal base pair on the 3'-5' exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3–6 s^–1 fast phase, 0.16–0.3 s^–1 slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04–0.22 s^–1), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3'-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition.

Received for publication, June 18, 2007 , and in revised form, March 7, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM 072705 (to E. T. K.) and GM 044613 (to K. A. J.). Conflict of interest: K. A. J. is president of KinTek Corp., which donated the RQF-3 rapid quench-flow instrument used in these studies. 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 Supported by Welch Foundation Grant F-1604. To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The 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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