We used norfloxacin to inhibit wild-type DNA gyrase and topoisomerase IV (Topo IV) in vivo as described elsewhere (27 (link),28 (link)). Drug-resistant mutants allowed us to selectively inhibit DNA gyrase or Topo IV. Cells were treated with 15 or 156 μM Norfloxacin for 15–30 min just before harvest. In all cases the DNA samples analyzed were prepared from separately isolated pools of supercoiled DNAs mixed in vitro with nicked forms of knotted or catenated molecules.
To induce single-stranded breaks, DNA was digested with Nb.BsmI (New England Biolabs) for 1 h at 8 U/μg of DNA at 50ºC. Reactions were blocked with 100 μg/ml proteinase K (Roche) for 30 min at 37ºC (29 (link)).
To induce single-stranded breaks, DNA was digested with Nb.BsmI (New England Biolabs) for 1 h at 8 U/μg of DNA at 50ºC. Reactions were blocked with 100 μg/ml proteinase K (Roche) for 30 min at 37ºC (29 (link)).
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