By Neil Osheroff, Mary-Ann Bjornsti
An extraordinary choice of state of the art experimental protocols for investigating the catalytic actions of DNA topoisomerases, in addition to their particular interactions with topoisomerase-targeted antitumor and antibacterial medicines. defined through specialist experimentalists who've perfected the recommendations, those unfailingly reproducible equipment contain assays for enzyme-catalyzed DNA relaxation/supercoiling, DNA cleavage, DNA nicking, DNA decatenation, and ATP hydrolysis. a number of transformed DNA substrates, used to dissect enzyme mechanisms by means of trapping intermediates, also are defined. Methodologies to figure out the motion of topoisomerase-targeted medications contain biochemical assays of drug-induced enzyme-DNA complexes, equipment for assaying drug uptake, and cell-based assays for deciding on the specificity and mechanisms of drug resistance. A spouse quantity, DNA Topoisomerase Protocols, I: DNA Topology and Enzymes, presents cutting-edge experimental protocols for investigating DNA constitution, topology, and DNA topoisomerase functionality.
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Additional resources for DNA Topoisomerase Protocols: Enzymology & Drugs
3. 4. 5. Haldane and Sullivan nuclear extract in 1–3 µl or 5–30 ng purified topoisomerase II). Bring to a total volume of 40 µL with H2O. Remember that topoisomerase II should be added last to start the reaction. To measure rates of decatenation at initial velocities use time-points of 15 s to 10 min. If the specific activity of the [3H]kDNA is too high, it can be diluted with cold kDNA. This reaction can be performed where either the concentration of topoisomerase II or the time of reaction is the variable.
0 mL of TE, and determine the DNA concentration by measuring the OD260 on a suitably diluted aliquot. 5 mg/500 mL bacterial culture. 8% agarose gel. It should be >90% supercoiled and should be stored at 4°C. The DNA is stable, but over a period of several months, may slowly undergo conversion to the nicked form. 2. Preparation of Relaxed pBR322 DNA The most convenient way of obtaining relaxed pBR322 is to relax the supercoiled form using a preparation of mammalian DNA topoisomerase I. , from calf thymus, chicken erythrocytes, or from the human HeLa cell line.
PB Sayer et al. Reverse Gyrase Activity 35 4 Analyzing Reverse Gyrase Activity Michel Duguet, Christine Jaxel, Anne-Cécile Déclais, Fabrice Confalonieri, Janine Marsault, Claire Bouthier de la Tour, Marc Nadal, and Christiane Portemer 1. Introduction In “inventing” reverse gyrase, the living world has built a unique and remarkably sophisticated enzyme that is more than a simple topoisomerase. Reverse gyrase possesses the unique ability to catalyze the production of positive supercoils in a closed-circular DNA at the expense of ATP (1); see ref.