Osheroff / Bjornsti DNA Topoisomerase Protocols

Volume II: Enzymology and Drugs
Erscheinungsjahr 2008
ISBN: 978-1-59259-057-5
Verlag: Humana Press
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 95, 334 Seiten, eBook

Reihe: Methods in Molecular Biology

Beginning with the Escherichia coli ? protein, or bacterial DNA topoisomerase I, an ever-increasing number of enzymes have been identified that catalyze changes in the linkage of DNA strands. DNA topoisomerases are ubiquitous in nature and have been shown to play critical roles in most p- cesses involving DNA, including DNA replication, transcription, and rec- bination. These enzymes further constitute the cellular targets of a number of clinically important antibacterial and anticancer agents. Thus, further studies of DNA topology and DNA topoisomerases are critical to advance our und- standing of the basic biological processes required for cell cycle progression, cell division, genomic stability, and development. In addition, these studies will continue to provide critical insights into the cytotoxic action of drugs that target DNA topoisomerases. Such mechanistic studies have already played an important role in the development and clinical application of antimicrobial and chemotherapeutic agents. The two volumes of DNA Topoisomerase Protocols are designed to help new and established researchers investigate all aspects of DNA topology and the function of these enzymes. The chapters are written by prominent investigators in the field and provide detailed background information and st- by-step experimental protocols. The topics covered in Part I: DNA Topology and Enzymes, range from detailed methods to analyze various aspects of DNA structure, from linking number, knotting/unknotting, site-specific recombi- tion, and decatenation to the overexpression and purification of bacterial and eukaryotic DNA topoisomerases from a variety of cell systems and tissues.

Zielgruppe

Professional/practitioner

Autoren/Hrsg.

Osheroff, Neil

Bjornsti, Mary-Ann

Inhaltsverzeichnis

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.