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J. Biol. Chem., Vol. 279, Issue 44, 45389-45398, October 29, 2004

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Effects of Primer-Template Sequence on ATP-dependent Removal of Chain-terminating Nucleotide Analogues by HIV-1 Reverse Transcriptase^*

Peter R. Meyer, Anthony J. Smith, Suzanne E. Matsuura, and Walter A. Scott

From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136-1015

HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP. However, the removal of 2',3'-dideoxy-TMP or 3'-azido-2',3'-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.

Received for publication, May 6, 2004 , and in revised form, July 21, 2004.

^* This work was supported by National Institutes of Health Grant R01 AI39973 (to W. A. S.), by American Foundation for AIDS Research Postdoctoral Fellowship 70567-31-RF (to P. R. M.), and by American Heart Association Predoctoral Fellowship 0215087B (to A. J. S.). 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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Miami School of Medicine, P.O. Box 016129, Miami, FL 33101-6129. Tel.: 305-243-6359; Fax: 305-243-3065; E-mail: wscott{at}med.miami.edu.

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