FIGURE 2.

TopA has nicking and closing activities. Plasmid pBR322 (1.77 nm) was incubated with purified topoisomerase I at the indicated concentrations for 1 h at 37 °C. The samples were processed and analyzed as described under “Experimental Procedures.” OC, relaxed open circles; CCC, covalently closed circles; RC, relaxed circular plasmids forms. Mw, molecular mass standard. A, gel run in the absence of EtBr. B, the same samples run in A were run in the presence of 0.5 μg/ml of EtBr. C, TopA activity on positively supercoiled pBR322. In wells labeled as (+) for EtBr, the pBR322 was incubated in the presence of 2 μg/ml of EtBr for 15 min at 4 °C before the incubation with TopA. In the well labeled (±), pBR322 previously treated with 2 μg/ml of EtBr was treated with isoamylalcohol to remove EtBr and further incubated with TopA. D, topoisomer distribution of pBR322 after TopA treatment. Plasmid DNA was subjected to two-dimensional agarose electrophoresis. E, illustration of pBR322 topoisomer distribution after treatment with 15 mm TopA in two-dimensional electrophoresis in agarose gels run in the presence of 1 and 2 μg/ml chloroquine in the first and second dimensions, respectively. Negative supercoiled topoisomers are in white, and positive supercoiled are in black. A black arrowhead indicates the topoisomer that migrated with ΔLk = 0 in the second dimension.