Figure 3.
ATPase and nuclease activities of St-Cas3 induced by St-Cascade binding to the proto-spacer dsDNA. (A) ATP hydrolysis rates. Malachite green assay was used to measure ATP hydrolysis through the detection of free phosphate liberated from ATP. Reaction rate constant k values (1/min) were calculated from linear slopes of time courses of phosphate liberation per St-Cas3 amount added. ATPase reactions were conducted at 37°C in the ATPase reaction buffer supplemented with 3 nM supercoiled plasmids, 12 nM St-Cascade and 300 nM St-Cas3 or the ATPase-deficient mutant D452A. Error bars indicate the ±standard deviation for the rate constant k value determined in three separate experiments. (B) dsDNA degradation requires St-Cas3, St-Cascade, and ATP. Nuclease reactions were conducted at 37°C for 10 min in a Nuclease buffer supplemented with 5 nM pSP1-AA and indicated amounts of St-Cas3 and St-Cascade. (C) PAM and a proto-spacer are essential for DNA degradation. Nuclease reactions were conducted at 37°C for indicated time intervals in Nuclease buffer supplemented with 100 nM St-Cas3, 20 nM St-Cascade, and 5 nM of respective supercoiled plasmids.
Source data for this figure is available on the online supplementary information page.
