Protocols Vol II

DNA Topoisomerase Protocols Volume II

Enzymology and Drugs

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.

Binding: Hardcover

Published: October 2000

Pages: 352 pp.

ISBN: 0-89603-512-3

Table of Contents

Assaying DNA Topoisomerase I Relaxation Activity, Lance Stewart and James J. Champoux. DNA Topoisomerase II-Catalyzed DNA Decatenation, Andrea Haldane and Daniel M. Sullivan. Plasmid DNA Supercoiling by DNA Gyrase, Penny J. Sayer, Martin L. Goble, Mark Oram, and L. Mark Fisher. Analyzing Reverse Gyrase Activity, Michel Duguet, Christine Jaxel, Anne-Céile Déclais, Fabrice Confalonieri, Janine Marsault, Claire Bouthier de la Tour, Marc Nadal, and Christiane Portemer. Topoisomerase II-Catalyzed ATP Hydrolosis as Monitored by Thin-Layer Chromatography, Paul S. Kingma, John M. Fortune, and Neil Osheroff. Use of a Real-Time, Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II, Janet E. Lindsley. Analysis of Topoisomerase–DNA Interactions by Electrophoretic Mobility Shift Assay, Stewart Shuman. Filter Binding Assays for Topoisomerase–DNA Complexes, Joaquim Roca. DNA Topoisomerase I-Mediated Nicking of Circular Duplex DNA, James J. Champoux. Analysis of Cleavage Complexes Using Reactive Inhibitor Derivatives, Kenneth N. Kreuzer, Catherine H. Freudenreich, and Yves Pommier. Uncoupling of Topoisomerase-Mediated DNA Cleavage and Religation, Anni H. Anderson, Kent Christiansen, and Ole Westergaard. Synthesis and Use of DNA Containing a 5'-Bridging Phosphorothioate as a Suicide Substrate for Type I DNA Topoisomerases, Alex B. Burgin, Jr. Isolation of Covalent Enzyme–DNA Complexes, Thomas C. Rowe, Dale Grabowski, and Ram Ganapathi. ICE Bioassay: Isolating In Vivo Complexes of Enzyme to DNA, Deepa Subramanian, Christine Sommer Furbee, and Mark T. Muller. DNA Unwinding Test Using Eukaryotic DNA Topoisomerase I, Linus L. Shen. Drug–DNA Interactions, David E. Graves. Quinolone Interactions with DNA and DNA Gyrase, Linus L. Shen. Bactericidal Assays for Fluoroquinolones, Thomas D. Gootz. Drug Toxicity in E. coli Cells Expressing Human DNA Topoisomerase I, Scott T. Taylor and Rolf Menzel. Drug-Induced Cytotoxicity in Tissue Culture, Cynthia E. Herzog and Leonard A. Zwelling. Analysis of Drug Uptake-Flow Cytometry, Grace Zoorob and Thomas Burke. Cell Cycle Analysis of Drug-Treated Cells, Frank Traganos, Gloria Juan, and Zbigniew Darzynkiewicz. Assaying Drug-Induced Apoptosis, Zbigniew Darzynkiewicz, Gloria Juan, and Frank Traganos. Single-Strand Conformational Polymorphism Analysis of DNA Topoisomerases I and II, Mary K. Danks. PCR-Based Cloning of DNA Topoisomerase Genes, Wai Mun Huang. Topoisomerase II-Catalyzed Relaxation and Catenation of Plasmid DNA, John M. Fortune and Neil Osheroff. Topoisomerase II-Mediated Cleavage of Plasmid DNA, D. Andrew Burden, Stacie J. Froelich-Ammon, and Neil Osheroff. Drug-induced Stabilization of Covalent DNA Topoisomerase I-DNA Intermediates: DNA Cleavage Assays, Paola Fiorani, Christine L. Hann, Piero Benedetti, and Mary-Ann Bjornsti. Studying DNA Topoisomerase I-Targeted Drugs in the Yeast Saccharomyces cerevisiae, Michael H. Woo, John R. Vance, and Mary-Ann Bjornsti. Yeast Systems for Demonstrating the Transfer of Antitopoisomerase II Agents, John L. Nitiss and Karen C. Nitiss. Index.

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