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J. Biol. Chem., Vol. 281, Issue 43, 32113-32121, October 27, 2006

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Reduced dNTP Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Promotes Strand Transfer^*

Darwin J. Operario¹, Mini Balakrishnan¹, Robert A. Bambara, and Baek Kim²

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

We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.

Received for publication, May 15, 2006 , and in revised form, August 17, 2006.

^* This work was supported in part by National Institutes of Health Research Grants AI49781 (to B. K.), GM49573 (to R. A. B.), and Fellowship AI056668 (to D. J. O.). 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 Both authors equally contributed to this work.

² 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; Email: baek_kim{at}urmc.rochester.edu.

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