By Neil Osheroff, Mary-Ann Bjornsti
Starting with the Escherichia coli ? protein, or bacterial DNA topoisomerase I, an ever-increasing variety of enzymes were pointed out that catalyze alterations within the linkage of DNA strands. DNA topoisomerases are ubiquitous in nature and feature been proven to play severe roles in such a lot p- cesses regarding DNA, together with DNA replication, transcription, and rec- bination. those enzymes additional represent the mobile objectives of a few clinically very important antibacterial and anticancer brokers. hence, additional experiences of DNA topology and DNA topoisomerases are severe to develop our und- status of the elemental organic strategies required for cellphone cycle development, cellphone department, genomic balance, and improvement. moreover, those experiences will proceed to supply serious insights into the cytotoxic motion of substances that focus on DNA topoisomerases. Such mechanistic stories have already performed an immense position within the improvement and medical program of antimicrobial and chemotherapeutic brokers. the 2 volumes of DNA Topoisomerase Protocols are designed to assist new and validated researchers examine all facets of DNA topology and the functionality of those enzymes. The chapters are written through favourite investigators within the box and supply distinct heritage info and st- by-step experimental protocols. the subjects coated partly I: DNA Topology and Enzymes, variety from exact tips on how to examine numerous facets of DNA constitution, from linking quantity, knotting/unknotting, site-specific recombi- tion, and decatenation to the overexpression and purification of bacterial and eukaryotic DNA topoisomerases from various cellphone structures and tissues.
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Extra resources for DNA Topoisomerase. Enzymology and Drugs Vol.II
2. Schematic model for positive supercoiling. The “hands” represent anchorage points of reverse gyrase to DNA required to maintain the separation between the two topological domains. activity contained in the C-terminal part would yield net positive supercoils (Fig. 2). Evidently, the composite structure of reverse gyrase that is described above results in multiple enzymatic activities and makes the analysis of its properties more difficult. , helicase) have not so far been demonstrated. This chapter will only describe the best-characterized activities of the enzyme.
1. General Description of the Assay The first reported use of this assay was by Kornberg and Pricer in 1951 (1). The assay makes use of the well-studied properties of the enzymes pyruvate kinase and lactate dehydrogenase, with the substrates MgADP, phospho(enol)pyruvate (PEP), and NADH. The reactions involved in the assay are illustrated in Scheme 1 below. All of the components, the ATPase (topoisomerase II in this case), MgATP (which will be referred to simply as ATP from here on), pyruvate kinase, lactate dehydrogenase, PEP, NADH, buffer, and salts, are mixed together and transferred to a cuvet.
9. Plastic-backed TLC plates are required if a scintillation counter is used to analyze samples. It is highly discouraged to attempt scraping and collecting radioactive resin from glass-backed plates. Acknowledgment This protocol was developed in part under the auspices of Grant GM33944 from the National Institutes of Health. References 1. , Zechiedrich, E. , and Gale, K. C. (1991) Catalytic function of DNA topoisomerase II. BioEssays 13, 269–273. 56 Kingma, Fortune, and Osheroff 2. Watt, P. M.