DNA Topoisomerase. Enzymology and Drugs Vol.II by Neil Osheroff, Mary-Ann Bjornsti

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 concerning DNA, together with DNA replication, transcription, and rec- bination. those enzymes extra represent the mobile ambitions of a few clinically vital antibacterial and anticancer brokers. hence, extra reviews of DNA topology and DNA topoisomerases are severe to enhance our und- status of the fundamental organic techniques required for cellphone cycle development, mobile department, genomic balance, and improvement. moreover, those reviews will proceed to supply serious insights into the cytotoxic motion of gear that concentrate on DNA topoisomerases. Such mechanistic stories have already performed an immense position within the improvement and scientific program of antimicrobial and chemotherapeutic brokers. the 2 volumes of DNA Topoisomerase Protocols are designed to aid new and confirmed researchers examine all features of DNA topology and the functionality of those enzymes. The chapters are written through famous investigators within the box and supply unique heritage info and st- by-step experimental protocols. the subjects lined partially I: DNA Topology and Enzymes, variety from special the way to research a variety of elements 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 numerous mobile platforms and tissues.

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It is important to draw lightly to ensure that the chromatographic resin is not scraped from the TLC plate. A representation of a TLC plate is shown in Fig. 1. 2. Prepare the assay buffer at room temperature (see Note 4) as follows (for a 20-µL reaction): mix 4 µL of 5X assay buffer stock, 1 µL of nonradioactive ATP (1 mM final concentration), appropriate volume of [γ-32P]ATP to contain ~10 µCi, appropriate volume of DNA to contain 200-µM base pairs (50 nM plasmid of pBR322) (see Note 5), and H2O so that the volume will be 20 µL following the addition of topoisomerase II (see Note 6).

In some cases, 4 mM MgCl2 are added to the reaction mixture. 2. 1 mM EDTA, 30 µg/mL bovine serum albumin, 10 mM MgCl2, 30 mM NaCl, and 6% ethylene glycol. 5 mg/mL proteinase K at 65°C in the presence of 1% SDS for 30 min, and two chloroform/isoamyl alcohol extractions. The aqueous phase is precipitated by ethanol in the presence of ammonium acetate, and the pellet is washed twice with ethanol 70%. 0, 1 mM EDTA (TE) buffer. 3. 4, 10 mM MgCl2, 20 mM NaCl. The total incubation volume is 100 µL, and the incubation is performed in the dark.

Lane 11 is a control without enzyme. 031 U (lane 10). Note that in processive conditions, the form I substrate (lower band in lanes 6–11) is still present for high amounts of reverse gyrase (lane 6), but individual positive topoisomers are already present in lane 6 and a high degree of positive supercoiling is obtained (lower bands in lanes 1–4). 1. The more commonly used condensing agent is polyethylene glycol (see Note 8). Addition of 7–9% PEG 6000 (purchased from Merck) to the reaction mixture in low salt (10–20 mM NaCl) yields highly positive DNA supercoils (16) (Fig.

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