MECHANISM OF QUINOLONE ACTION - A DRUG-INDUCED STRUCTURAL PERTURBATION OF THE DNA PRECEDES STRAND CLEAVAGE BY TOPOISOMERASE-IV

Quinolones are potent broad spectrum antibacterial drugs that target t he bacterial type II DNA topoisomerases, Their cytotoxicity derives fr om their ability to shift the cleavage-religation equilibrium required for topoisomerase action toward cleavage, thereby effectively trappin g the enzyme on the DNA. It has been proposed that these drugs act by binding to the enzyme-DNA complex. Using catalytically inactive and qu inolone-resistant mutant topoisomerase IV proteins, nitrocellulose fil ter DNA binding assays, and KMnO4 probing of drug-DNA and drug-DNA-enz yme complexes, we show: (i) that norfloxacin binding to DNA induces a structural alteration, which probably corresponds to an unwinding of t he helix, that is exacerbated by binding of the topoisomerase and by b inding of the drug to the enzyme and (ii) that formation of this struc tural perturbation in the DNA precedes DNA cleavage by the topoisomera se in the ternary complex. We conclude that cleavage of the DNA and th e resultant opening of the DNA gate during topoisomerization requires the induction of strain in the DNA that is bound to the enzyme. We sug gest that quinolones may act to accelerate the rate of DNA cleavage by stimulating acquisition of this structural perturbation in the ternar y complex.