Mechanistic role of residue Gln151 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'-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.template/primer) during DNA polymerization. We also found that by changing residue 151 from a Gln-->Asn, the maximum rate of dNTP incorporation (k(pol)) for both correct and incorrect dNTPs was not affected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs (K(d)) was diminished. 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 were unable to measure the actual binding affinity of Q151N for incorrect dNTPs. 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 Gln(151) is important for tight binding of incorrect dNTPs and may contribute to the low fidelity nature of HIV-1 RT. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Gln(151) residue may play an important role in efficient binding of RT to correct dNTPs. Our findings suggest that residue Gln(151) is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.
Weiss
Kellie K
KK
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Bambara
Robert A
RA
Kim
Baek
B
D004247:DNA / Q000096:biosynthesis / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry* / Q000502:physiology; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D015151:Immunoblotting; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014633:Valine / Q000737:chemistry
D005973:Glutamine; D004247:DNA; D054303:HIV Reverse Transcriptase; D014633:Valine