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Papers In Press, published online ahead of print April 10, 2002
J. Biol. Chem, 10.1074/jbc.M200282200
Submitted on January 10, 2002
Revised on March 25, 2002
Accepted on April 9, 2002
Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
Corresponding Author: prasad{at}aecom.yu.edu
HIV-1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template-primer contacts have been shown to influence its fidelity and sensitivity to nucleoside-analog inhibitors. The Phe61 residue, located within the b3 sheet of the fingers subdomain, is highly conserved among retroviral RTs. Crystal structure of a ternary complex revealed that Phe61 contacts the first and second bases of the 5’-template overhang. In order to determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (PheÆAla, Leu, Trp or Tyr) at Phe61. The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe61 mutant RTs also displayed large reductions in sensitivity to 2’,3’-dideoxythymidine triphosphate (ddTTP) and 2’,3’-dideoxy, 2’3’-didehydrothymidine triphosphate (d4TTP). Mutants displaying the largest increase in fidelity (F61A, F61L) were also the most resistant. These results suggest that contacts between the fingers subdomain of HIV-1 RT and the template 5’-overhang are important determinants of the geometry of the dNTP-binding pocket.
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