| Binding properties of the human immunodeficiency virus (HIV) reverse transcriptase were examined. The RNase H activity of HIV-RT is coordinated by a catalytic triad (E478, D443, D498) of acidic residues that bind divalent cations. The effects of two RNase H deficient single point mutants (E 478 > Q and D549 > N) that do not alter polymerase activity on binding of enzyme to various nucleic acid substrates were examined. Binding of the mutant and wild type enzymes to various nucleic acid substrates was examined by determining dissociation rates (koff ) and dissociation equilibrium constants (Kd) as a function of both Mg2+ and salt concentrations. In agreement with the unaltered polymerase activity of the mutant, the k off values for the wild type and mutant enzymes were essentially identical using DNA-DNA templates in the presence of 6 mM Mg2+. However, with lower Mg2+ concentrations and in its absence, although both enzymes dissociated more rapidly, the mutant enzymes dissociated several fold more slowly than the wild type. This was also observed on RNA-DNA templates. These results indicate that alterations in residues essential for Mg 2+ binding have a pronounced positive effect on enzyme-template stability, and that the negatively charged residues in the RNase H region of the enzyme have a negative influence on binding of RT to its primer-templates in the absence of Mg2+. In addition, the wild type-enzyme may have two distinct or mutually exclusive Mg2+ binding sites, a low affinity and a high affinity site, both of which serve to stabilize the binding of the enzyme to the primer template. In this regard RT is similar to other nucleic acid cleaving enzymes that show enhanced binding upon mutation of negatively charged active site residues.; The role of sequence specificity in RT binding to DNA-DNA primer-template structures was examined by using a modified version of the SELEX (s&barbelow;ystematic e&barbelow;volution of l&barbelow;igands by exponential enrichment) protocol. A series of random duplex DNA-DNA sequences with 3′ recessed termini were generated and subjected to several rounds of selection via a gel-shift assay in an attempt to identify and isolate sequences with high affinity. Isolated substrates were then assayed to determine the dissociation rates (koff) and dissociation equilibrium constants (Kd) of the substrate HIV-RT complexes. After ten rounds of selection, substrates with lower dissociation rates and dissociation equilibrium constants were isolated and analyzed. Ramifications and importance of these data are discussed, specifically regarding the importance of this data in drug design. |
