Fig 3.

Analysis of efficiency of processive DNA synthesis. (A) Comparative analysis of the enzyme efficiency of processive DNA synthesis of HIV-1 WT RT and mutant RT at high dNTP concentrations (200 μM). The D25 primer labeled with ³²P at the 5′ end (³²P-D25) was annealed to the 471-nt HIV-1 PBS RNA template, and primer extension assays were performed at an excess of recombinant RT enzymes at high dNTP concentrations (200 μM). Reactions were stopped at 0.4 min, 1 min, and 6 min. The longest extension products generated at 1 min are identified by arrows and indicate differences in the efficiency of polymerization. Each experiment was repeated at least twice and yielded similar results on each occasion. The figure shows a gel from a representative experiment. (B) Comparative analysis of enzyme efficiency of processive DNA synthesis of HIV-1 WT RT and mutant RT at low dNTP concentrations (0.5 μM). Reactions were stopped at 2 min and 4 min. The longest extension products generated at 4 min are identified by arrows and indicate differences in the efficiency of polymerization. Each experiment was repeated at least twice and yielded similar results on each occasion. The figure shows a gel from a representative experiment.