Figure 4

Unwinding of blunt-ended duplex substrates by WRN-E84A and BLM. A) Helicase reactions containing 2 mM ATP, fully duplex (labeled C80/G0) substrate (0.5 nM) and WRN-E84A (96 nM) were incubated at 37°C, with or without addition of unlabeled C80 (0.5–12.5 nM) at 3 min into the incubation. Reactions were terminated after 15 min total and DNA products analyzed as described in Methods. Quantitation of the amount of WRN-dependent unwinding is presented above lanes 2–6. B) Parallel helicase reactions were performed as in A using WRN-E84A (96 nM) with or without unlabeled C80 (5 nM) added 3 min into the reaction. For each reaction, 0, 7.5, 15, and 30 min time points were taken and DNA products analyzed as above. Quantitation of the level of unwinding in reactions with or without unlabeled C80 appears at right. C) Parallel helicase reactions containing labeled C80/G80 (0.05 nM), BLM (12 nM) with or without unlabeled C80 (0.2 nM) were performed, analyzed, and depicted graphically as in B. D) Parallel helicase reactions containing fully duplex substrate (0.5 nM) and WRN-E84A (77 nM) were incubated at 37°C with or without addition of SDS (1%) at 7.5 min into the incubation. Time points taken at 0, 7.5, 15, and 30 min were analyzed as above. Quantitation of the percentage of unwound product over time appears at right. E) Time points (0, 7.5, 15, and 30 min) from parallel helicase reactions are analyzed and depicted as in D, except using UvrD (14 nM) instead of WRN-E84A.