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 DNA 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 play a frontline role in cancer biology as factors that can both maintain and disrupt genome integrity; they are also demonstrated drug targets for treating cancer. Our past research on eukaryotic topoisomerase II (topo II) has opened up new research avenues for understanding cancer etiology and improving cancer treatment. The present application will deliver groundbreaking solutions to key problems in the field, including how certain classes of anti-topo II drugs act on the enzyme, how topo II is localized to key sites of action where it resolves potentially deleterious chromosomal topologies, and how aberrant topo II activity can promote DNA damage and genetic instability. We will also investigate innovative concepts and highly significant lines of inquiry raised by our new findings, such as how metabolites produced by the TCA cycle control topo II function. Our approach is distinguished by a comprehensive blend of biochemical, structural, computational, cell-based, and chemical biology methodologies. High-impact outcomes will include defining how topo II appropriately localizes with chromatin and partner proteins to mitigate its natural DNA-damaging potential, establishing how the specificity of anti-topo II agents can be improved to enhance their utility in cancer treatment, and revealing the potential for natural amino-acid sequence variation in type II topoisomerases to destabilize human chromosomes and act as cancer drivers. Past progress and unpublished findings establish the feasibility of our planned goals.