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. 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 cytofoxic 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 Volume 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.

Klappentext

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.

Inhalt
to DNA Topoisomerases.- Resolution of DNA Molecules by One-Dimensional Agarose-Gel Electrophoresis.- Two-Dimensional Agarose-Gel Electrophoresis of DNA Topoisomers.- Analysis of Altered DNA Structures.- Purification of Supercoiled Plasmid DNA.- Purification and Use of DNA Minicircles with Different Linking Numbers.- Isolation of Kinetoplast DNA.- Isolation of Knotted DNA from Coliphage P4.- Analysis of DNA Knots and Catenanes by Agarose-Gel Electrophoresis.- Sedimentation Analysis of Bacterial Nucleoid Structure.- Coating DNA with RecA Protein to Distinguish DNA Path by Electron Microscopy.- Methods for Analyzing DNA Bending.- Formation of Extrachromosomal DNA Rings in Saccharomyces cerevisiae Using Site-Specific Recombination.- Overexpression and Purification of Bacterial DNA Gyrase.- Overexpression and Purification of Bacterial DNA Topoisomerase I.- Overexpression and Purification of Escherichia coliDNA Topoisomerase III.- Overexpression and Purification of Bacterial Topoisomerase IV.- Purification of the Bacteriophage T4 Type II DNA Topoisomerase.- Overexpression and Purification of DNA Topoisomerase I from Yeast.- Overexpression and Purification of Saccharomyces cerevisiae DNA Topoisomerase II from Yeast.- Purification of DNA Topoisomerase II from Drosophila melanogaster.- Purification of DNA Topoisomerase I from Human Placenta.- Purification of Baculovirus-Expressed Human DNA Topoisomerase I.- Rapid Purification of DNA Topoisomerase II Containing a Hexahistidine Tag by Metal Ion Affinity Chromatography.- Metabolic Labeling, Immunoprecipitation, and Two-Dimensional Tryptic Phosphopeptide Mapping of Human Topoisomerase II.- Immunoblot Analysis and Band Depletion Assays.- Visualization of DNA Topoisomerases by Electron Microscopy.- Appendix: Compendium of DNA Topoisomerase Sequences.