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J. Biol. Chem., Vol. 281, Issue 38, 27873-27881, September 22, 2006

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Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference^*

Chandravanu Dash, Timothy S. Fisher¹², Vinayaka R. Prasad², and Stuart F. J. Le Grice³

From the Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702 and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.

Received for publication, April 25, 2006 , and in revised form, June 28, 2006.

^* This work was supported in part by the Intramural Research Program of the National Institutes of Health, NCI, and Center for Cancer Research (to C. D. and S. Le G.). 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 Present address: Division of Cardiovascular Diseases, Merck Research Laboratories, Rathway, NJ 07065.

² Supported by Public Health Service Research Grant AI 30861.

³ To whom correspondence should be addressed. Tel.: 301-846-5256; Fax: 301-846-6013; E-mail: slegrice{at}ncifcrf.gov.

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