The persistence of topoisomerase IV-DNA cleavage complexes established in the presence of drugs was determined using the procedure of Gentry et al. (46 (link)). Initial reaction mixtures contained 1 μM wild-type or mutant topoisomerase IV, 50 nM DNA, and 20 μM (for the wild-type enzyme) or 200 μM (for the mutant enzymes) ciprofloxacin or 20 μM 8-methyl-quinazoline-2,4-dione in a total of 20 μL of DNA cleavage buffer. Reactions were incubated at 37 °C for 10 min and then diluted 20-fold with DNA cleavage buffer warmed to 37 °C. Samples (20 μL) were removed at times ranging from 0-300 min, and DNA cleavage was stopped with 2 μL of 5% SDS followed by 1 μL of 250 mM EDTA (pH 8.0). Samples were digested with proteinase K and processed as described above for plasmid cleavage assays. Levels of DNA cleavage were set to 100% at time zero, and the persistence of cleavage complexes was determined by the decay of the linear reaction product over time.