Faculty Scholarship

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 Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV.

Results: In 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 1mM 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 significance: At 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.

 DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have thefluoroquinolone C-7ring systemfacing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modifiedchloroacetylderivativeofciprofloxacin(CipAcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTAandthermaltreatments.Surprisingly,cross-linking was also readily seen with complexes formed by mutant GyrAG81Cgyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinoloneresistant GyrA-G81C variant of Escherichia coli and its Mycobacteriumsmegmatisequivalent(GyrA-G89C).Cip-AcClmediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinoloneC-7ringandbothGyrAandGyrB.BecausetheGyrAGly81andGyrB-Glu466residuesarefarapart(17Å)inthecrystal structureofcleavedcomplexes,twomodesofquinolonebinding mustexist. The presence of two binding modesraises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNAtopoisomerases.