Figure 9.

DNA pre-bound at all four sites in the PaqCI tetramer is cut sequentially. (A) Cleavage of a four-site plasmid substrate either pre-bound (left) or not pre-bound (right) with a 1:1 enzyme-binding-site to DNA-sites ratio. The pre-bound reaction (left gel) contained 37 nM DNA target sites and 37 nM of PaqCI per 37 nM DNA target sites in 50 μl buffer lacking Mg²⁺ ions. The enzyme was allowed to bind for 15 min at 37°C. An aliquot was removed (time 0) and the cleavage reaction was initiated by adding Mg²⁺ to 10 mM. The no pre-binding reaction (right gel) 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, 0.5, 1, 3, 5, 10, 30, 60 min. The mobilities of the super coil (SC), single-cut linear (Linear), intermediates (brackets), and the completely-cleaved linear (frag1, frag2, frag3 and frag4) forms of the plasmid are indicated in the middle key with arrows corresponding to their related bands. Intermediate bands correspond to a less than fully cleaved plasmid; single, dual, triple and complete cleavage events. To the left of the gels is a cartoon of the substrate and reaction products shown in the gel. Molecular weight ladders were Lambda-HindIII/PhiX174_HaeIII which spans 0.2–23.1 kb. These experiments were performed once. (B) Same reaction conditions and plasmid, as in Panel a right side, 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. These experiments were performed once.