FIGURE 2.

Dna2 cannot track on a primed circular long single strand without a free 5′-end. Helicase activity was assayed as described under “Experimental Procedures.” Reactions containing 15 fmol of either (A) M13mp18 containing a completely annealed primer (M13mp18: D4) or (B) M13mp18 containing a primer with an unannealed 5′-flap (M13mp18:D5) were incubated with 300 fmol of Dna2K677R for 30 min at 37 °C. The reaction buffer contained 2 mm ATP and 2 mm Mg Cl₂. Lanes 1 and 5 are substrate-alone controls, and lanes 2 and 6 are boiled substrate controls. Lanes 3 and 7 contained only 2 mm MgCl₂ in the reaction buffer. Lanes 4 and 8 contained 2 mm ATP and 2 mm MgCl₂ in the reaction buffer. The asterisk on the substrates depicted above the figures denotes the position of the radiolabel. Positions of the helicase products are indicated in the figure.