Fig. 4.
HIV-1 RT can use HPMPCpp or HPMPApp to support DNA synthesis. (A and B) Primer extension reactions examining the ability of HIV-1 RT to replace dCTP with HPMPCpp. FAM-labeled primer P1 was annealed to template T9RNA or T9 (A) or to template T2RNA or T2 (B) and incubated at 37°C with 50 nM HIV-1 RT in the presence of 10 μM (each) dATP, dGTP, and dTTP. Reactions were further supplemented with 10 μM ddCTP (lanes 2 and 9), 10 μM dCTP (lanes 3 and 10), or 10 μM HPMPCpp (lanes 4 to 7 and 11 to 14). ddCTP- and dCTP-containing reaction mixtures were incubated for 20 min; HPMPCpp-containing reaction mixtures were incubated over 20 min with sampling at 1, 5, 10, and 20 min. (C and D) Primer extension reactions examining the ability of HIV-1 RT to replace dATP with HPMPApp. Reaction mixtures were prepared by annealing FAM-labeled primer P1 to template T11RNA or T11 (C) or to template T12RNA or T12 (D) and incubated at 37°C with 50 nM HIV-1 RT in the presence of 10 μM (each) dCTP, dGTP, and dTTP. ddATP at 10 μM (lanes 2 and 9), dATP at 10 μM (lanes 3 and 10), or HPMPApp at 10 μM (lanes 4 to 7 and 11 to 14) was also added as indicated. ddATP- and dATP-containing reaction mixtures were incubated for 20 min; HPMPApp-containing reaction mixtures were incubated over 20 min, with sampling at 1, 5, 10, and 20 min. Lanes marked “Primer” show labeled primer P1. The locations of pause sites are indicated by arrows.