Fig. 7.

Compound 2 does not inhibit ADPNP-induced relaxation of positively supercoiled DNA by E. coli DNA gyrase. Reactions containing gyrase, the indicated compounds (at 4 µM) ± nucleotide were carried out with positively supercoiled DNA (see Materials and Methods for details). (Upper) After relaxation, cleavage complexes were removed from the DNA by sequential addition of EDTA and proteinase K to the reaction (this procedure fully reverses the cleavage induced by both compounds—tested independently on relaxed DNA) (SI Appendix, Fig. S7). Rel, relaxed DNA, +SC, positively supercoiled DNA. (Lower) After relaxation, cleavage complexes were trapped on the DNA by the addition of SDS to identical reactions as in Upper. The gel in Lower was run in the presence of 0.5 µg/mL ethidium bromide (see Materials and Methods for details). Asterisk denotes multiple cleavage events resulted in the smearing of the DNA substrate on the gel.