FIGURE 4.

Degradation of target DNA requires both the PAM and seed sequences. A, extent of nicking by Cas3 (100 nm) using negatively supercoiled target (2 nm) containing the indicated point mutations in the PAM and protospacer. All reactions contained 10 mm Mg²⁺ and 10 μm Co²⁺. B, rate of ATP hydrolysis by Cas3 (100 nm) in the presence of the mutant DNA targets as in A. In both panels, error bars indicate S.D., taken from at least three independent measurements. ΔPAM, DNA target containing a protospacer sequence but not a PAM.