FIGURE 7.

Specificity of RPA stimulation of WRN branch migration activity. A, RPA stimulation of WRN on D-loops lacking a tail. The 5′ no tail mobile non-telomeric D-loop (38) (50 nm) was incubated with 1.2 nm X-WRN and increasing concentrations of RPA (0, 0.62, 1.9, 5, or 5 nm) (lanes 1–5, respectively) for 15 min under standard reaction conditions. The reactions were run on a 4–20% native gel. B, quantitation of the reactions in A were calculated as described under “Experimental Procedures” and plotted against RPA concentration. Values represent the mean and S.D. from two or three independent reactions. C and D, the telomeric mobile D-loop (50 pm) was incubated with either 5 nm X-WRN and increasing concentrations of T4 single strand binding protein (0, 2.5, 7.5, 20, or 60 nm) (C) or 1.2 nm RecQ and increasing concentrations of RPA (0, 0.62, 1.9, 5, or 15 nm) (D). Reactions were conducted under standard conditions for 15 min, run on a 4–20% native polyacrylamide gel, and visualized by PhosphorImager analysis. ▲, heat-denatured substrate. The percentage of total ssDNA displacement (%TD) was calculated as described under “Experimental Procedures.”