ABSTRACT The appropriate control of DNA topology has a major impact on the stability and flow of genetic information. The present application focuses on type II topoisomerases, molecular machines that modulate DNA supercoiling and remove chromosome entanglements by catalyzing the ATP-dependent transport of one DNA duplex through another. Type II topoisomerases are critical for maintaining gene expression, chromosome superstructure, and genome integrity; they also serve as frontline drug targets for treating infectious disease and cancer. During the prior project period, we gained several new insights into the mechanism, regulation, and DNA damage propensity of type II topoisomerases. These efforts open up three new research opportunities centered on complementary but non-interdependent aspects of type II topoisomerase action that will advance both fundamental biological knowledge and therapeutic intervention. Aim 1 seeks to explain the long-unresolved question of how type II topoisomerases link ATP-dependent allosteric responses to the selective engagement and release of multiple DNA segments, and to illuminate how a system responsible for initiating meiotic recombination co-opted and modified a topoisomerase scaffold to generate DNA breaks for promoting chromosome exchange. Aim 2 will break new ground in understanding the regulation of eukaryotic topo II, uncovering natural metabolites that control enzyme activity as means to modulate replication, transcription, and chromosome organization processes that are sensitive to DNA topology. Aim 3 will determine how the action of human topo IIβ drives aberrant DNA damaging events and how naturally-occurring mutations may further potentiate this detrimental activity. Our approach is distinguished by a comprehensive blend of biochemical, structural, computational, cell-based, and chemical biology methodologies. Past progress and unpublished findings provide data to establish feasibility for the proposed effort. Our studies will impact multiple fields, from the study of molecular machines and the control of DNA dynamics, to understanding how topoisomerase activity and its regulation support specific physiological needs and promote human reproduction and health.