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

Background Fluoroquinolones 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. Methods We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg²⁺-, Mn²⁺-, or Ca²⁺-supported DNA cleavage activity of Escherichia coli Topo IV. Results In the absence of any drug, 20-30 mM Mg²⁺ was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn²⁺ or Ca²⁺ 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 Mg²⁺ concentrations where Topo IV alone could not efficiently cleave DNA. Conclusions and general significance At low Mg²⁺ concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg²⁺ binding to metal binding site B through the structural distortion in DNA. As Mg²⁺ concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg²⁺ at site B or inhibition the binding of Mg²⁺ to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg²⁺ binding.