Assaying DNA Topoisomerase I Relaxation Activity -- DNA Topoisomerase II-Catalyzed DNA Decatenation -- Plasmid DNA Supercoiling by DNA Gyrase -- Analyzing Reverse Gyrase Activity -- Topoisomerase II-Catalyzed ATP Hydrolysis as Monitored by Thin-Layer Chromatography -- Use of a Real-Time, Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II -- Analysis of Topoisomerase-DNA Interactions by Electrophoretic Mobility Shift Assay -- Filter Binding Assays for Topoisomerase-DNA Complexes -- DNATopoisomerase I-Mediated Nicking of Circular Duplex DNA -- Analysis of Cleavage Complexes Using Reactive Inhibitor Derivatives -- Uncoupling of Topoisomerase-Mediated DNA Cleavage and Religation -- Synthesis and Use of DNA Containing a 5?-Bridging Phosphorothioate as a Suicide Substrate for Type I DNA Topoisomerases -- Isolation of Covalent Enzyme-DNA Complexes -- ICE Bioassay -- DNA-Unwinding Test Using Eukaryotic DNA Topoisomerase I -- Drug-DNA Interactions -- Quinolone Interactions with DNA and DNA Gyrase -- Bactericidal Assays for Fluoroquinolones -- Drug Toxicity in E. coli Cells Expressing Human Topoisomerase I -- Drug-Induced Cytotoxicity in Tissue Culture -- Analysis of Drug Uptake-Flow Cytometry -- Cell-Cycle Analysis of Drug-Treated Cells -- Assaying Drug-Induced Apoptosis -- Single-Strand Conformational Polymorphism Analysis of DNA Topoisomerases I and II -- PCR-Based Cloning of DNA Topoisomerase Genes -- Topoisomerase II-Catalyzed Relaxation and Catenation of Plasmid DNA -- Topoisomerase II-Mediated Cleavage of Plasmid DNA -- Drug-induced Stabilization of Covalent DNA Topoisomerase I-DNA Intermediates -- Studying DNATopoisomerase I-Targeted Drugs In the Yeast -- Yeast Systems for Demonstrating the Targets of Anti-Topoisomerase II Agents.

DNA Topoisomerase Protocols, II: Enzymology and Drugs brings together 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 drug specificity and mechanisms of 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. DNA Topoisomerase Protocols, II: Enzymology and Drugs provides, for the first time, a comprehensive description of the methods and experimental systems needed to investigate fully the catalytic mechanisms of DNA topoisomerases from bacterial, viral, and eukaryotic sources. For the many basic scientists and clinicians who want better to understand and to exploit the action of topoisomerase-targeted drugs in a variety of in vitro and in vivo systems, it will immediately develop immense value as the new standard reference in the field.