Figure 3.

Type I-A Cascade cleaves ssDNA unspecifically and is inhibited by crRNA. (A) Cascade (0, 0.05, 0.125, 0.25, 0.5 µM) was incubated with a 5′-[γ-³²P]-ATP labeled ssDNA fragment (int_5.2 CCT for) in the presence of Mg²⁺ and Mn²⁺ ions and the nucleolytic cleavage reaction was resolved on 15% denaturing gels. After 1–10-min incubation at a fixed Cascade concentration of 0.1 µM, >70% of the substrate is cleaved. In the presence of 0.5 µM unlabeled crRNA, the reaction is inhibited. (B) This observation was tested in the presence of 0, 0.05, 0.5 and 2.5 µM crRNA, and the amount of remaining substrate estimated via line profile plots (Image J) was plotted for three different reaction times (1, 2, 10 min) and reactions performed in triplicate. (C) Cas3′′ subunit with HD domain mutations (H19A, H55A, D56A) was assembled into Cascade and then tested in ssDNA cleavage assays.