Isolation and quantitation of topoisomerase complexes accumulated on Escherichia coli chromosomal DNA

DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabi-lizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound fromDNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, al-lowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin con-centrations greater than 1MIC.When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1 and 1.7MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrAmutation on accumulation of gyrase covalent complexes on chromosomal DNA. DNA topoisomerases have been important targets for the dis-covery of anticancer and antibacterial drugs (3, 24, 26, 34, 35). The drugs that target topoisomerases currently used in ther-apy are effective because they act as topoisomerase poisons by stabilizing the covalent topoisomerase-DNA complex intermedi-ates (6, 22, 27, 28). Methods for detection and quantitation of endogenous topoisomerase-DNA covalent complexes formed on chromosomal DNA were developed for mammalian cells (4, 14