Figure 6. Kinetics of WRN activity on plasmid D-loops.

Reactions contained 50 pM of either the 5′Tail, No Tail, or 3′Tail plasmid D-loops as indicated. The 5′ labeled end is indicated with a star. The substrate was incubated with 5 nM WRN (Panel A–B) or 5 nM E84A-WRN mutant (Panel C) for 0.5, 1, 2, 4, 8, 15, 30 min under standard reaction conditions. Reactions were run on 4–20% polyacrylamide native gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. B. and C. The percent total displacement was calculated as described in Materials and Methods and plotted against time for reactions with wild type WRN (B.) or the E84A-WRN mutant (C.). 5′Tail D-loop, ▪ and black line; 3′Tail D-loop, ▴ and blue line; No Tail D-loop, ▾and yellow line. The representative phosphorimage scans for the E84A-WRN reactions are shown in Supplemental Fig. S3A.