FIG. 7.

Ciprofloxacin and its symmetric dimer (dimer 2) induce unwinding of closed circular DNA. Relaxed plasmid pBR322 (0.8 μg) was incubated with calf thymus DNA topoisomerase I (12 U) in the presence of 10 mM MgCl₂ at room temperature for 10 min before addition of ciprofloxacin (CIP) and dimer 2 at the indicated concentrations. After incubation for 1 h at 37°C, the topoisomerase I was inactivated and the DNA was extracted with phenol to remove any bound ligand prior to agarose gel electrophoresis in Tris-phosphate-EDTA buffer. Half of each DNA sample was run in the absence of chloroquine (−CQ); the other half was electrophoresed in the presence of chloroquine (+CQ). Under the conditions of the gel without chloroquine, all the DNA samples are positively supercoiled and are made more positively coiled by the inclusion of chloroquine. Lanes a, pBR322 DNA (previously relaxed in the absence of Mg²⁺ ions), used as the substrate.