Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site

BackgroundFluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.MethodsWe conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg^2 +-, Mn^2 +-, or Ca^2 +-supported DNA cleavage activity of Escherichia coli Topo IV.ResultsIn the absence of any drug, 20–30 mM Mg^2 + was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn^2 + or Ca^2 + was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg^2 + concentrations where Topo IV alone could not efficiently cleave DNA.Conclusions and general significanceAt low Mg^2 + concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg^2 + binding to metal binding site B through the structural distortion in DNA. As Mg^2 + concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg^2 + at site B or inhibition the binding of Mg^2 + to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg^2 + binding.