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J. Biol. Chem., Vol. 283, Issue 21, 14402-14410, May 23, 2008

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Importance of Hydrogen Bonding for Efficiency and Specificity of 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 the Department of Chemistry, Stanford University, Stanford, California 94305

To assess the contribution to discrimination afforded by base pair hydrogen bonding during DNA replication by the human mitochondrial DNA polymerase, we examined nucleoside mimics lacking hydrogen bond forming capability but retaining the overall steric shape of the natural nucleotide. We employed oligonucleotide templates containing either a deoxyadenosine shape mimic (dQ) or a deoxythymidine shape mimic (dF). Additionally, the nucleoside triphosphate analogs difluorotoluene deoxynucleoside triphosphate, 9-methyl-1-H-imidazo[(4,5)-b]pyridine deoxyribose triphosphate, and 4-methylbenzimidazole deoxyribose triphosphate (dZTP; another dATP shape mimic) were assayed. We used pre-steady state methods to determine the kinetic parameters governing nucleotide incorporation, k_pol and K_d. In general, the loss of hydrogen bonding potential led to 2–3 kcal/mol reduction in ground state binding free energy, whereas effects on the maximum rate of polymerization were quite variable, ranging from negligible (dATP:dF) to nearly 4 kcal/mol (dZTP:dT). Although we observed only a 46-fold reduction in discrimination when dF was present in the template, there was a complete elimination of discrimination when dQ was present in the template. Our data with dF indicate that hydrogen bonding contributes 2.2 kcal/mol toward the efficiency of incorporation, whereas data with dQ (which may overestimate the effect due to poor steric mimicry) suggest a contribution of up to 6.8 kcal/mol. Taken together, the data suggest that sterics are necessary but not sufficient to achieve optimal efficiency and fidelity for DNA polymerase. Base pair hydrogen bonding contributes at least a third of the energy underlying nucleoside incorporation efficiency and specificity.

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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				H. R Lee, S. A. Helquist, E. T. Kool, and K. A. Johnson Base Pair Hydrogen Bonds Are Essential for Proofreading Selectivity by the Human Mitochondrial DNA Polymerase J. Biol. Chem., May 23, 2008; 283(21): 14411 - 14416. [Abstract] [Full Text] [PDF]