Figure 3.

Cleavage activity of I-DmoI and I-DmoI_CL. (A) Typical time-course activity assay. The 7 min assay was performed with pUD718 plasmid dsDNA and I-DmoI (top) or I-DmoI_CL (bottom); 20 µl samples were removed at the times indicated. Closed-circular plasmid DNA (C) was cleaved forming a linear product (L); nicked, open circles (O) appeared as intermediates of the reaction. Residual nuclease activity was ruled out by incubating each construct with target plasmid lacking only the 14 bp recognition sequence. No nicking or cleavage activity was observed (G. H. Silva, unpublished results). (B) Plots of data in (A). Percent reaction products are shown over time. Diamonds, squares and triangles correspond to O, L and C, respectively, in (A).