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Papers In Press, published online ahead of print April 1, 2002
J. Biol. Chem, 10.1074/jbc.M200202200
Submitted on January 8, 2002
Revised on March 28, 2002
Accepted on March 30, 2002
Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642
Corresponding Author: baek_kim{at}urmc.rochester.edu
We previously reported that mutations in residue Q151 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Q151 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 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 Q151 residue is not involved in formation of the binary complex (RT-T/P) during DNA polymerization. We also found that by changing residue 151 from a GlnàAsn, the maximum rate of dNTP incorporation (kpol) for both correct and incorrect dNTPs was not affected. In contrast, the ability of this 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 decreased binding to incorrect dNTPs was such that we were unable to measure the actual binding affinity of Q151N. 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 Q151 is important for tight binding of incorrect dNTPs and may contribute to the HIV-1 RT infidelity. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Q151 residue may be important in efficient binding of RT to correct dNTPs. Our findings suggest that residue Q151 is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.
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