DNA Topoisomerase Protocols

1. Front Matter

   Pages i-xvi

   PDF

2. Assaying DNA Topoisomerase I Relaxation Activity

   • Lance Stewart, James J. Champoux

   Pages 1-11

3. DNA Topoisomerase II-Catalyzed DNA Decatenation

   • Andrea Haldane, Daniel M. Sullivan

   Pages 13-23

4. Plasmid DNA Supercoiling by DNA Gyrase

   • Penny J. Sayer, Martin L. Goble, Mark Oram, L. Mark

   Pages 25-33

5. Analyzing Reverse Gyrase Activity

   • Michel Duguet, Christine Jaxel, AnneCécile Déclais, Fabrice Confalonieri, Janine Marsault, Claire Bouthier de la Tour et al.

   Pages 35-49

6. Topoisomerase II-Catalyzed ATP Hydrolysis as Monitored by Thin-Layer Chromatography

   • Paul S. Kingma, John M. Fortune, Neil Osheroff

   Pages 51-56

7. Use of a Real-Time, Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II

   • Janet E. Lindsley

   Pages 57-64

8. Analysis of Topoisomerase-DNA Interactions by Electrophoretic Mobility Shift Assay

   • Stewart Shuman

   Pages 65-74

9. Filter Binding Assays for Topoisomerase—DNA Complexes

   • Joaquim Roca

   Pages 75-80

10. DNATopoisomerase I-Mediated Nicking of Circular Duplex DNA

    • James J. Champoux

    Pages 81-87

11. Analysis of Cleavage Complexes Using Reactive Inhibitor Derivatives

    • Kenneth N. Kreuzer, Catherine H. Freudenreich, Yves Pommier

    Pages 89-99

12. Uncoupling of Topoisomerase-Mediated DNA Cleavage and Religation

    • Anni H. Andersen, Kent Christiansen, Ole Westergaard

    Pages 101-117

13. Synthesis and Use of DNA Containing a 5′-Bridging Phosphorothioate as a Suicide Substrate for Type I DNA Topoisomerases

    • Alex B. Burgin Jr.

    Pages 119-128

14. Isolation of Covalent Enzyme-DNA Complexes

    • Thomas C. Rowe, Dale Grabowski, Ram Ganapathi

    Pages 129-135

15. ICE Bioassay

    • Deepa Subramanian, Christine Sommer Furbee, Mark T. Muller

    Pages 137-147

16. DNA-Unwinding Test Using Eukaryotic DNA Topoisomerase I

    • Linus L. Shen

    Pages 149-160

17. Drug—DNA Interactions

    • David E. Graves

    Pages 161-169

18. Quinolone Interactions with DNA and DNA Gyrase

    • Linus L. Shen

    Pages 171-184

19. Bactericidal Assays for Fluoroquinolones

    • Thomas D. Gootz

    Pages 185-194

20. Drug Toxicity in E. coli Cells Expressing Human Topoisomerase I

    • Scott T. Taylor, Rolf Menzel

    Pages 195-204

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

• Vanderbilt University School of Medicine, Nashville

  Neil Osheroff

• St. Jude Children’s Research Hospital, Memphis

  Mary-Ann Bjornsti

Policies and ethics