An unprecedented collection of cutting-edge experimental protocols for investigating the catalytic activities of DNA topoisomerases, as well as their specific interactions with topoisomerase-targeted antitumor and antibacterial drugs. Described by expert experimentalists who have perfected the techniques, these unfailingly reproducible methods include assays for enzyme-catalyzed DNA relaxation/supercoiling, DNA cleavage, DNA nicking, DNA decatenation, and ATP hydrolysis. Several modified DNA substrates, used to dissect enzyme mechanisms by trapping intermediates, are also described. Methodologies to determine the action of topoisomerase-targeted drugs include biochemical assays of drug-induced enzyme-DNA complexes, methods for assaying drug uptake, and cell-based assays for determining the specificity and mechanisms of drug resistance. A companion volume, DNA Topoisomerase Protocols, I: DNA Topology and Enzymes, provides state-of-the-art experimental protocols for investigating DNA structure, topology, and DNA topoisomerase function.