FIGURE 5.

Comparison of supercoiling extent by wild-type and insert-less E. coli gyrase. A, negative supercoiling activity. Protein concentrations are listed in nm holoenzyme, and asterisks denote the minimal concentrations of enzyme needed to supercoil the substrate in 30 min. B, negative supercoiling time course assay using 20 nm holoenzyme. Time points are listed in minutes. A portion of each sample was run on a 1% agarose gel in the absence (44) and presence (bottom) of 3 μg/ml chloroquine. DNA topoisomers are depicted with graphic representations at the right side of the native gel, and the topological state indicated as relaxed (Rel.) or negatively supercoiled DNA species ((-) sc) at the bottom of the chloroquine gel. All reactions were started with 6 nm relaxed pSG483 plasmid DNA substrate.