DNA Topoisomerase Protocols

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.

"This book completes a comprehensive collection of assays, including the weird and wonderful, that can be used to investigate the enzymology and drug inhibition of topoisomerases...I recommend any students about to undertake a PhD degree in this area to buy a copy. Even those who are well versed in the biology of topoisomerases should consider buying both volumes of this book, they will surely find some things they didn't know - I did!" - MicroBiology Today

"Described by expert experimentalists who have perfected the techniques, these reproducible methods include assays for enzyme-catalyzed DNA relaxation/supercoiling, DNA cleavage, DNA nicking, DNA decatentaion, and ATP hydrolysis." - Anticancer Research

"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." -Tumori

Klappentext

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.

Inhalt
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 TopoisomeraseDNA 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.- DrugDNA 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.