Figure 4.

Irreversibility of CL-stimulated topoisomerase IV-mediated DNA cleavage sites. A 5′ end ³²P-labelled 239 bp-long SV40 DNA segment was incubated with topoisomerase IV and 25 µM of CPF (lanes 3–5) or 100 µM of CL (lanes 6–8) at 37°C for 20 min. Samples in lanes 4 and 7, and 5 and 8 were further treated with 0.6 M NaCl for 20 min (indicated as RS) or 65°C for 20 min (indicated as RT), respectively. Stopped reaction mixtures were loaded and run on a 8% denaturing polyacrylamide gel. DNA fragments were visualized by phosphorimaging. Lane 1 and 2 are controls for untreated DNA (D) and topoisomerase IV-processed DNA, respectively.