Project Summary Topoisomerase II poisons represent some of the most widely prescribed and efficacious anticancer and antibacte- rials drugs; yet, currently, anticancer drugs can lead to cardiotoxicity and secondary malignancies, and the growing prevalence of drug-resistant bacteria threatens the efficacy of antibacterial drugs. A key step towards designing new, more effective, and less harmful drugs is understanding the molecular mechanisms of topoisomerase II poisons. These drugs target type II topoisomerases, essential enzymes that relax the topological and mechanical stress that builds up on DNA during transcription, replication, and recombination. As part of these enzymes' reaction cycle, they bind at crossings between two DNA segments, reversibly cleave one DNA segment, generating a transient protein-DNA cleavage complex, and pass the other segment through the resulting gap. Most drug-based poisons act as “molecular doorstops” and prevent religation of the cleaved DNA segment. As a result, enzyme activity is inhibited and topological and mechanical stress builds up in cells; however the effects of this stress on the poison's mechanism of action is unknown. A leading hypothesis is that the buildup of this stress further enhances poison binding, increasing the lifetime of cleavage complexes and inhibiting enzyme activity, as well as converting cleavage complexes to permanent DNA damage. However, recapitulating this stress in experiments is a major obstacle in the field. To address this obstacle, I propose to use high-precision single-molecule force spectroscopy experiments to recapitulate physiologically relevant levels of topological and mechanical stress and study its effects on poison binding and cleavage complex rupture with various poison-based drugs. These experiments will provide unprecedented mechanistic insight into the effects of stress and poisons that is likely to be important for the efficacy of these drugs and help guide the development of new drugs.