Figure 8 Time-dependent digestion of plasmids with four PaqCI target sites indicate a sequential cleavage at individual sites

Panel a. Cleavage of a head-to-head four-site substrate with and without an activator added in trans. The no activator reaction contained 1 ug substrate DNA (37 nM sites) and 2 units (17.2 nM) of PaqCI per ug DNA in 50 ul rCutSmart buffer at 37C. The activator reaction had the same substrate and PaqCI enzyme together with 80 nM activating oligoduplex (40 nM per unit PaqCI) in 50 ul rCutSmart buffer at 37C. Digest time points were quenched at 0.25 minute (min), 0.5 min, 1 min, 3 min, 5 min, 10 min, 30 min, and 60 minutes. This experiment was performed once. Panel b. The cleavage of a head-to-tail four-site substrate with and without an activator added in trans. Reactions were conducted as in Panel a. This experiment was performed once.