Unraveling the molecular mechanism of type II topoisomerase poisons with magnetic tweezers.

Abstract

Topoisomerases are an essential class of enzymes that resolve topological problems on DNA (e.g., supercoils, knots, and links) that arise during transcription, replication, and recombination. As part of their reaction cycle, type II topoisomerases transiently cleave double-stranded DNA and form a covalent protein-DNA cleavage complex. Typically, these cleavage complexes are tightly regulated in vivo because they are highly vulnerable and can generate cytotoxic DNA breaks, yet some of the most efficacious and broad-spectrum antibacterials are topoisomerase poisons that exploit this vulnerability by disrupting cleavage complex regulation. Due to their biological and clinical importance, the effects of these poisons have been studied extensively with a variety of in vitro ensemble assays. These assays have shown that antibacterials increase overall levels of cleavage complexes and inhibit enzyme activity. However, the kinetics of individual poisoned cleavage complexes under physiologically relevant conditions is unknown. Therefore, we carried out single-molecule magnetic tweezer measurements of E. coli topo IV activity on supercoiled DNA. In the presence of topoisomerase poisons, we observed long pauses in enzyme activity that correspond to poison-stabilized cleavage complexes. We studied the effects of DNA tension, as well as supercoil handedness and extent, determining poison on-rates, cleavage-complex lifetimes, and topoisomerase activity with several common antibacterials under physiologically relevant conditions. Together, these results provide new mechanistic insight into how topoisomerase poisons disrupt cleavage complex regulation that is likely to be relevant to their action in vivo.