DNA Topoisomerases Protocols, Volume 2: Enzymology and Drugs

About this book

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.

Contents

Overview Neil Osheroff and Mary-Ann Bjornsti. Assaying DNA Topoisomerase I Relaxation Activity Lance Stewart and James J. Champoux. Assaying DNA Topoisomerase II- Catalyzed DNA Relaxation and Catenation Neil Osheroff. DNA Topoisomerase II Catalyzed DNA Decatenation Andrea Haldane and Daniel Sullivan. Plasmid DNA Supercoiling by DNA Gyrase Penny Sayer Martin L. Goble Mark Oram and L. Mark Fisher. Analyzing Reverse Gyrase Activity Michel Duguet Christine Jaxel Anne Ceile Declais Fabrice Confalonieri Janine Marsualt Claire Bouthier de la Tour Marc Nadel and Christine Portemer. Topoisomerase II-Catalyzed ATP Hydrolysis as Monitored by Thin-Layer Chromatography Paul S. Kingma John M. Fortune and Neil Osheroff. Use of Real-Time Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II Janet E Lindsley. Analysis of Topoisomerase-DNA Interactions by Electrophoretic Mobility Band 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. DNA Topoisomerase II-Mediated Plasmid DNA Cleavage Neil Osheroff. DNA Topoisomerase I-Induced Cleavage of Linear DNA Substrates Mary-Ann Bjornsti. Analysis of Cleavage Complexes Using Reactive Inhibitor Derivatives Kenneth N. Kreuzer Catherine H. Freudenreich and Yves98-015 Pommier. DNA Topoisomerase II-Induced Cleavage of Linear DNA Substrates and Analysis of Cleavage Sites 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. Burgen Jr. DNA Topoisomerase II-Mediated Religation of DNA Neil Osheroff. Isolation of Covalent Enzyme-DNA Complexes Thomas C. Rowe Dale Grabowski and Ram Ganapathi. Alkaline and Neutral Elution Kurt Kohn. ICE Bioassay: Isolating In Vivo Complexes of Enzyme to DNA Deepa Subramanian Christine Sommer Furbee and Mark T. Muller. Drug Unwinding Test Using Eukaryotic DNA Topoisomerase I Linus L. Shen. Drug-DNA Interactions David 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. Establishing the Specificity of DNA Topoisomerase-Targetted Drugs in Yeast John Nitiss and Mary-Ann Bjornsti. Selection of Drug-Resistant DNA Topoisomerase I Mutants: Yeast Mary-Ann Bjornsti and Piero Benedetti. Selection of Drug-Resistant DNA Topoisomerase II Mutants: Yeast John Nitiss. Drug-Induced Cytotoxicity in Tissue Culture Cynthia E. Herzog and Leonard A. Zwelling. Analysis of Drug Uptake-Flow Cytometry Grace K. Zoorob and Thomas G. Burke. Cell-Cycle Analysis of Drug-Treated Cells Frank Traganos Gloria Juan and Zeigniew Darzynkiewicz. Assaying Drug-Induced Apoptosis Zeigniew Darzynkiewicz Gloria Juan and Frank Traganos. Selection of Drug-Resistant Mutants: Mammalian Cells William Beck. Single- Strand Conformational Polymorphism Analysis of DNA Topoisomerase I and II Mary K. Danks. PCR-Based Cloning of DNA Topoisomerase Genes Wai-Mun Huang. Future Goals James C. Wang.

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