Fluoroquinolone-dependent DNA supercoiling by Vaccinia topoisomerase I

Vaccinia topoisomerase I is a site-specific DNA strand transferase that acts through a DNA-(3'-phosphotyrosyl)-enzyme intermediate, resulting in relaxation of supercoiled DNA. Although Vaccinia topoisomerase I is not an essential enzyme, its role in early transcription makes it a potential antiviral target. We describe the interaction of Vaccinia topoisomerase I with fluoroquinolone antibiotics otherwise known to target DNA gyrase and topoisomerase IV in bacterial cells. The fluoroquinolone enrofloxacin inhibits DNA relaxation by Vaccinia topoisomerase I at concentrations similar to those required for inhibition by the coumarin drugs coumermycin and novobiocin. When Vaccinia topoisomerase I is presented with relaxed DNA in the presence of enrofloxacin, it executes the reverse reaction, supercoiling the DNA. Further characterization indicates that enrofloxacin does not interfere with the initial strand scission by Vaccinia topoisomerase I. The structurally related fluoroquinolones moxifloxacin and lomefloxacin have no effect on the topoisomerase at the concentrations at which enrofloxacin mediates DNA supercoiling. The mechanism with which Vaccinia topoisomerase I supercoils relaxed DNA, an energetically unfavorable, yet ATP-independent process, must entail protein-DNA contacts downstream of the cleavage site, as opposed to the free rotation mechanism proposed for DNA relaxation; as proposed for fluoroquinolone-mediated inhibition of gyrase, the drug may target a preformed topoisomerase I-DNA complex to induce conformational changes in the enzyme that permit such contacts.