Reduced dNTP interaction of human immunodeficiency virus type 1 reverse transcriptase promotes strand transfer

We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.     

Mismatched and matched dNTP incorporation by DNA polymerase beta proceed via analogous kinetic pathways

While matched nucleotide incorporation by DNA polymerase beta (Pol beta) has been well-studied, a true understanding of polymerase fidelity requires comparison of both matched and mismatched dNTP incorporation pathways. Here we examine the mechanism of misincorporation for wild-type (WT) Pol beta and an error-prone I260Q variant using stopped-flow fluorescence assays and steady-state fluorescence spectroscopy. In stopped-flow, a biphasic fluorescence trace is observed for both enzymes during mismatched dNTP incorporation. The fluorescence transitions are in the same direction as that observed for matched dNTP, albeit with lower amplitude. Assignments of the fast and slow fluorescence phases are designated to the same mechanistic steps previously determined for matched dNTP incorporation. For both WT and I260Q mismatched dNTP incorporation, the rate of the fast phase, reflecting subdomain closing, is comparable to that induced by correct dNTP. Pre-steady-state kinetic evaluation reveals that both enzymes display similar correct dNTP insertion profiles, and the lower fidelity intrinsic to the I260Q mutant results from enhanced efficiency of mismatched incorporation. Notably, in comparison to WT, I260Q demonstrates enhanced intensity of fluorescence emission upon mismatched ternary complex formation. Both kinetic and steady-state fluorescence data suggest that relaxed discrimination against incorrect dNTP by I260Q is a consequence of a loss in ability to destabilize the mismatched ternary complex. Overall, our results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol beta originates from destabilization of the mismatched closed ternary complex and chemical transition state.     

Role of glutamine 151 of human immunodeficiency virus type-1 reverse transcriptase in substrate selection as assessed by site-directed mutagenesis

A natural mutation at codon 151 (Gln --> Met; Q151M) of HIV-1 RT has been shown to confer resistance to the virus against dideoxy nucleoside analogues [Shirasaka, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2398], suggesting that Gln 151 may be involved in conferring sensitivity to nucleoside analogues. To understand its functional implication, we generated two mutant derivatives of this residue (Q151M and Q151N) and examined their sensitivities to ddNTPs and their ability to discriminate against rNTPs versus dNTP substrates on natural U5-PBS HIV-1 RNA template. We found that Q151M was highly discriminatory against all four ddNTPs but was able to incorporate rNTPs as efficiently as the wild type enzyme. In contrast, the Q151N mutant was only moderately resistant to ddNTPs but exhibited a higher level of discrimination against rNTPs. The fidelity of misinsertion was found to be highest for the Q151N mutant followed by Q151M and the wild type enzyme. These results point toward the importance of the amino acid side chain at position 151 in influencing the ability of the enzyme in recognition and discrimination against the sugar moieties of nucleotide substrates.