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Originally published In Press as doi:10.1074/jbc.M200202200 on April 1, 2002

J. Biol. Chem., Vol. 277, Issue 25, 22662-22669, June 21, 2002
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Mechanistic Role of Residue Gln151 in Error Prone DNA Synthesis by Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase (RT)
PRE-STEADY STATE KINETIC STUDY OF THE Q151N HIV-1 RT MUTANT WITH INCREASED FIDELITY*

Kellie K. WeissDagger , Robert A. Bambara§, and Baek KimDagger

From the Departments of Dagger  Microbiology and Immunology and § Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

It has previously been reported that mutations in the Gln151 residue of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Gln151 in highly error prone DNA synthesis by HIV-1 RT. Using our Q151N high fidelity mutant, which is structurally altered in its ability to interact with the 3'-OH on the sugar moiety of the incoming deoxynucleotide triphosphate (dNTP), we examined how this change in RT-dNTP interaction affects HIV-1 RT fidelity. First, we found the binding affinity (KD) of wild type and Q151N RT proteins to different template/primers to be similar. These results indicate that the Gln151 residue is not involved in the formation of the binary complex (RT·template/primer) during DNA polymerization. We also found that by changing residue 151 from a Glnright-arrowAsn, the maximum rate of dNTP incorporation (kpol) for both correct and incorrect dNTPs was not affected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs (Kd) was diminished. The Q151N mutant was 120-fold less efficient at binding correct dNTP than wild type RT, and its decrease in binding was such that we were unable to measure the actual binding affinity of Q151N for incorrect dNTPs. Presumably, the fidelity increase observed during the steady state is explained by this defect in Q151N binding to incorrect dNTP. In wild type RT, residue Gln151 is important for tight binding of incorrect dNTPs and may contribute to the low fidelity nature of HIV-1 RT. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Gln151 residue may play an important role in efficient binding of RT to correct dNTPs. Our findings suggest that residue Gln151 is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.

* This work was supported by Grants GM55500 (to B. K.) and GM29573 (to R. A. B.) and Training Grant AI07362-12 (to K. K. W.) from the National Institutes of Health.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.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642. Tel.: 585-275-6916; Fax: 585-473-9573; E-mail: baek_kim@urmc.rochester.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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