Figure 5.

CL-stimulated DNA cleavage in the presence of wild-type topoisomerase IV or mutant quinolone-resistant enzyme reconstituted with ParC S79F. A 5′ end ³²P-labelled 239 bp-long SV40 DNA segment was incubated with wild-type (lanes 2, 4–5, 8–9) or S79F mutant topoisomerase IV (lanes 3, 6–7, 10–11) and 25–50 µM of CPF (lanes 4–7) or CL (lanes 8–11) at 37°C for 30 min. Reaction mixtures were stopped and run on a 8% denaturing polyacrylamide gel. DNA fragments were visualized by phosphorimaging. Lane 1 is a control for the untreated input DNA. Lanes 2–3 are controls for wild-type and S79F mutant topoisomerase IV-processed DNA, respectively. Arrows on the right of the gel indicate cleavage bands and asterisks specify cleavage sites that are differently induced by CL-stimulated wild-type and S79F mutant enzymes.