Figure 9 DNA pre-bound at all four sites in the PaqCI tetramer is cut sequentially

Panel a. Cleavage of a four-site plasmid substrate either pre-bound or not pre-bound with a 1:1 enzyme-binding-site to DNA-sites ratio. The pre-bound reaction contained 1 ug substrate DNA (37 nM sites) and 4.3 units (37 nM) of PaqCI per ug DNA per 50 ul rCutSmart buffer lacking Mg++ ions and enzyme was allowed to bind for 15 minutes at 37C. An aliquot was removed (time 0) and the cleavage reaction was initiated by adding Mg++ to 10 mM. The no pre-binding reaction had the same substrate and PaqCI enzyme concentrations in normal buffer, with time points started upon enzyme addition. 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. Panel b. Same reaction conditions as in panel a, except that the PaqCI enzyme concentration was increased to 2:1 (74 nM enzyme to 37 nM DNA sites) or 4:1 (148 nM enzyme to 37 nM DNA sites) ratio of enzyme to DNA sites.