ATP-mediated excision of AZTMP from RNA/DNA template–primers by WT and mutant RTs. Assays were carried out with 31/21-mer RNA–DNA complexes (sequences shown below the upper left panel). The inhibitor was first incorporated at position +1 (indicated with an asterisk) of the 21-nucleotide primer (lane P) to generate a 22-nucleotide product (lane B). 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 a fully extended 31-nucleotide product. Aliquots were removed 2, 4, 6, 8, 10, 12, 15, and 20 min after the addition of ATP. Time courses of the excision reactions are shown in three panels, each one containing data from different sets of related RTs. Time courses obtained with WT HIV-2 RT and mutant 5M are included in all panels for comparison. All dNTPs in the assays were supplied at 200 μm, except for dATP whose concentration was 2 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.