FIGURE 2.

Determination of the dissociation rate constants of the HIV-1 RTs. A, a typical dissociate rate experiment is shown. The substrate used was a 24-nt DNA primer annealed to a 41-nt RNA template. Experiments were performed in the presence of excess trap, oligo(dT)-poly(rA). The time axis indicates how long after the oligo(dT)-poly(rA) trap was added until the Mg²⁺ and dNTPs were added to initiate DNA synthesis. The amount of the extended products was proportional to the amount of RT remaining bound at each time point. Lane C represents a control reaction in which the trap was added before the RT. B, the amount of primer extension seen at increasing times of incubation with the trap is shown relative to the amount of primer extension when there was no preincubation with the trap. Nonlinear least-square fits of the data from three independent experiments to an equation for single-exponential decay are shown. C, the dissociation rate constants were calculated for the three HIV-1 RTs.