DNA Topoisomerase. Enzymology and Drugs Vol.II

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. 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 DNA Topoisomerase Protocols is an unprecedented collection of cutting-edge experimental protocols for investigating DNA structure, topology, and DNA topoisomerase function. Described by expert experimentalists who have perfected the techniques, these time-proven methods utilize new approaches to study changes in DNA topology (linking number, knotting, catenation, and relaxation) and DNA structure (bending). as well as to assess chromosome structure through FLP-mediated recombination and to analyze bacterial nucleoid structure. State-of-the-art protocols also detail the expression and purification of bacterial, viral, and eukaryotic DNA topoisomerases from a variety of sources, including baculovirus, bacterial, yeast, and mammalian expression systems Volume II: Enzymology and Drugs contains detailed methodologies for the analysis of enzyme activities, including the relaxation of supercoiied DNA, DNA unknotting and decatenation, ATP hydrolysis, and the formation of drugstabilized, covalent-enzymc DNA intermediates. Additional chapters describe methods for analyzing topoisomerase-iargeted drug action. A variety of experimental ystems are presented to assess the cytotoxic action opoisomerase-directed agents, as well as methods for selecting and analyzing drug-resistant DNA topoisomerase mutants. Beginning with the Escherichia coli co protein, or bacterial DNA topoisomerase I, an ever-increasing number of enzymes has been identified that catalyze changes in the linkage of DNA strands. Type I topoisomerases catalyze topological changes in duplex DNA by reversibly nicking one strand, whereas type II enzymes catalyze the transient breakage of both strands simultaneously. Pt. 2. Enzymology And Drugs Edited By Neil Osheroff And Mary-ann Bjornsti. Includes Bibliographical References And Index.