Mechanistic Role of Residue Gln 151 in Error Prone DNA Synthesis by Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase (RT) PRE-STEADY STATE KINETIC STUDY OF THE Q151N HIV-1 RT MUTANT WITH INCREASED FIDELITY*

It has previously been reported that mutations in the Gln 151 residue of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Gln 151 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 (cid:1) -OH on the sugar moiety of the incoming deoxynucleotide triphosphate (dNTP), we examined how this change in RT-dNTP interaction affects HIV-1 RT fidelity. First, we found the binding affinity ( K D ) of wild type and Q151N RT proteins to different template/primers to be similar. These results indicate that the Gln 151 residue is not involved in the formation of the binary complex (RT (cid:2) template/primer) during DNA polymerization. We also found that by changing residue 151 from a Gln 3 Asn, the maximum rate of dNTP incorporation ( k pol ) for both correct and incorrect dNTPs was not af- fected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs ( K d ) was dimin- ished. The Q151N mutant was 120-fold less efficient at binding correct dNTP than wild type RT, and its decrease in binding was such that we The state single experiments were RT was used in slight excess T/P In reactions involving incorrect dNTPs, the experiments were performed manually at longer periods of time and used a higher concentration of RT (700 n M ) (15, 26). Product Analysis— The reactions were analyzed by 14% denaturing sequencing gel electrophoresis. The extended product in each reaction was quantified with the Cyclone phosphorimager (PerkinElmer Life Sciences). Data Analysis— Pre-steady state kinetic data were analyzed using nonlinear regression. Equations were generated with the KaleidaGraph program, version 3.51 (Synergy Software). Data points obtained during the burst experiment were fitted to the burst equation shown below 11).