Figure 4.

ATP-mediated excision of nucleoside analogues from RNA/DNA and DNA/DNA template–primers by WT HIV-2 RT and mutants M73K, D67N/K70R, and D67N/K70R/M73K. Assays were carried out with 31/21-mer RNA–DNA complexes (a) or 38/25-mer DNA–DNA complexes (b and c) (sequences shown above excision reaction time courses). The inhibitors (AZT in a and b, and tenofovir in c) were first incorporated at position +1 (indicated by asterisks) to generate 22- or 26-nucleotide products. Excision of the inhibitor and further primer extension in the presence of 3.2 mm ATP and a mixture of dNTPs led to the formation of fully extended primers of 31 or 38 nucleotides. Aliquots were removed at the appropriate times after ATP addition. Time courses of the excision reactions are shown below. The dNTPs in the assays carried out with RNA–DNA complexes were supplied at 200 μm, except for dATP whose concentration was 2 μm. In assays using DNA–DNA complexes, the dNTP concentration was kept at 100 μm, except for dATP (AZT-terminated primers) or dTTP (tenofovir-terminated primers) which was supplied at 1 μm. Template–primer and active RT concentrations in these assays were 30 and 24 nm, respectively. Represented values (means ± S.D. error bars) were obtained from at least three independent experiments.