Fig 2.

Comparative analysis of enzyme processivity of WT RT and RT enzymes containing the indicated substitutions. The processivity of purified recombinant RT enzymes was analyzed using a 5′-end-labeled DNA primer (D25) annealed to a 471-nt HIV-1 PBS RNA template as the substrate; the resulting full-length DNA is 471 nt in length. Processivities were determined by the size distribution of DNA products in fixed-time experiments at low (0.5 μM) and high (200 μM) concentrations of dNTPs in the presence of a heparin trap. All reaction products were resolved by denaturing 6% polyacrylamide gel electrophoresis and visualized by phosphorimaging. The positions of the ³²P-labeled D25 primer (³²P-D25) and the 471-nt full-length (FL) extension DNA product are indicated on the right. Lanes: 1, WT; 2, M184I mutant; 3, K101E/M184I mutant; 4, E138K/M184I mutant; 5, K101E/E138K/M184I mutant; 6, K101E mutant; 7, K101E/E138K mutant. The results of a control reaction to verify the efficiency of the heparin trap by preincubation with substrate prior to addition of WT RT are also shown (lane C). Each experiment was repeated at least twice and yielded similar results on each occasion. The figure shows a gel from a representative experiment.