Figure 4

PAT produces covalently closed, relaxed DNA. (A) Lane 1 contains the 1 Kb ladder, lane 2 contains 120 ng of supercoiled DNA, lane 3 contains 130 ng nicked DNA, lane 4 contains 140 ng linear DNA and lane 5 contains 140 ng supercoiled pUC18 and 63 ng protein in topoisomerase buffer with MgCl₂. The reaction mixtures were run on 1% agarose gel. (B) Lane 1 contains the 1 Kb ladder, lane 2 contains 120 ng supercoiled DNA, lane 3 contains 130 ng nicked DNA, lane 4 contains 140 ng linear DNA, lane 5 contains 140 ng supercoiled pUC18 and 63 ng protein in topoisomerase buffer with MgCl₂. The reaction mixtures were run on a 1% agarose gel with 2 μg/ml ethidium bromide. Samples in panels A and B represent the same reaction, half of which is loaded on each gel. For reactions in lanes 5, quantitation of relative amounts of supercoiled DNA shows that <8% of total DNA remains supercoiled after incubation with topoisomerase (panel (A)), while ~35% of total DNA is supercoiled after electrophoresis in the presence of ethidium bromide (panel (B)), indicating that ~30% of total DNA is covalently closed following reaction with PAT. Panels (C) and (D) are equivalent to (A) and (B) with lanes 2–5 containing supercoiled, linear, nicked, and relaxed DNA, respectively; DNA in lanes 5 is relaxed with Vaccinia topoisomerase IB.