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. 2014 Feb 4;289(18):12300–12312. doi: 10.1074/jbc.M113.529164

FIGURE 3.

FIGURE 3.

Evidence for cross-linking of GyrA-G81C gyrase to haloacetyl derivatives of ciprofloxacin. Reaction mixtures containing gyrase or topoisomerase IV, supercoiled pBR322 DNA, and derivatives of ciprofloxacin were incubated at 37 °C as described under “Experimental Procedures.” A–C, display of plasmid DNA species following incubation with derivatives of ciprofloxacin and purified gyrase or topoisomerase IV. Concentrations of enzymes and drugs were chosen to obtain similar amounts of linear DNA. The DNA cleavage assay used 5 nm plasmid pBR322 DNA and 7.5 nm wild-type gyrase (A), 25 nm GyrA-G81C gyrase (B), or 7.5 nm topoisomerase IV (C). Ciprofloxacin was used at 1 μm for wild-type gyrase; it was 25 μm for GyrA-G81C gyrase and topoisomerase IV. Cip-Ac, Cip-AcBr, and Cip-AcCl were used at 5 μm for wild-type gyrase and 125 μm for GyrA-G81C gyrase and topoisomerase IV. S and E indicate DNA products from reaction mixtures without and with 50 mm EDTA-mediated reversal, respectively. The assay was repeated twice with identical results (less than 2% difference in the percentage of linear DNA). The effect of drug concentration on the recovery of linear DNA is shown in D–F; data were obtained as in Fig. 2 except that GyrA-G81C was substituted for GyrB-E466C. For the data shown, concentrations of enzyme and DNA were 20 and 7.4 nm, respectively; reactions were incubated for 30 min at 37 °C and then treated either with 50 mm EDTA and then SDS (empty symbols) or with SDS first and then EDTA (filled symbols). D shows the effect of the cross-linking agent Cip-AcCl with GyrA-G81C gyrase, E shows results for non-cross-linking ciprofloxacin with GyrA-G81C gyrase (circles) and for non-cross-linking Cip-Ac with GyrA-G81C gyrase (triangles), and F shows results for wild-type gyrase with the cross-linking agent Cip-AcCl. Examination of a variety of enzyme:DNA ratios and incubation times produced results similar to those shown. The number of total experiments and those used for the data shown (chosen so panels had the same incubation conditions), respectively, were 9 and 4 for the comparison of D, 10 and 2 for ciprofloxacin in E, 8 and 4 for Cip-Ac in E, and 7 and 2 for wild-type gyrase and the cross-linking agent Cip-AcCl in F. Total DNA recovery was unaffected by drug concentration, indicating that loss of specific bands was not due to preferential recovery. Error bars indicate S.D.