Figure 4.

Mapping the cleavage events on the bottom strand following rear-end collision at a site. (A) Cartoon of the linear DNA substrate from Figure 3. The sequence of the LlaGI site is highlighted, along with the locations cleaved by LlaBIII. The sun symbol shows the labeling with 32-phosphorus. (B) DNA (2 nM), 5′-labeled on the bottom strand with 32-phosphorus (sun symbols), was incubated with the enzymes shown (200 nM of each) for 2 min. ATP was added to 4 mM, the reaction incubated for 10 min at 25°C, and then stopped. The products were separated by denaturing polyacrylamide gel electrophoresis. (C) Quantified band intensities for lanes 6–13 (in grey) in panel B (Materials and Methods). Bar represents 500 intensity units. Tables key: No enzyme (−); WT enzyme (+ or WT); nuclease point mutant (N−); nuclease domain deletion (ΔN); or, ATPase point mutant (H−). Gel and quantitation shown are representative examples from two repeat reactions.