By Neil Osheroff, Mary-Ann Bjornsti
An exceptional number 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 medicinal drugs. defined by way of specialist experimentalists who've perfected the concepts, those unfailingly reproducible tools contain assays for enzyme-catalyzed DNA relaxation/supercoiling, DNA cleavage, DNA nicking, DNA decatenation, and ATP hydrolysis. numerous changed DNA substrates, used to dissect enzyme mechanisms by way of trapping intermediates, also are defined. Methodologies to figure out the motion of topoisomerase-targeted medicines comprise 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, offers state of the art experimental protocols for investigating DNA constitution, topology, and DNA topoisomerase functionality.
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Extra resources for DNA Topoisomerase Protocols: Enzymology & Drugs
9, 3589–3603. 13. , Gormley, N. , Smith, C. , Fisher, L. , and Duncan, K. (1996) The mode of action of GR122222X, a novel inhibitor of DNA gyrase. Antimicrob. Agents Chemother. 40, 473–476. 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.
PBR322 is commercially available, is readily propagated in E. coli, and is a substrate for gyrase from a variety of sources. The following sections describe the isolation of supercoiled pBR322, preparation of relaxed pBR322 substrate, and its use in the supercoiling assay. 2. 1. Isolation of Supercoiled pBR322 from E. coli 1. 5 with 5 M NaOH. For solid media, add 15 g/L of agar. Autoclave for 20 min at 15 lb/sq. in. 2-µ filter (Nalgene) is added where appropriate to 50 µg/mL. 2. 0, containing 50 mM glucose and 10 mM NaEDTA.
Add an equal volume of 6% N-lauroylsarcosine, and swirl to mix. 5 × 108/mL is equal to 9 × 1010 total trypanosomes. This will require a total combined volume of buffer 1 and 6% N-lauroylsarcosine of 90 mL to lyse at 1 × 109/mL; 45 mL of each is added). 6. 5 mL of 20 mg/mL proteinase K). Gently mix and place in a 37°C water bath for 30 min. 7. , Beckman 25 × 89 mm tubes, cat. no. 344058) place 24 mL of solution A. 0 mL of solution B to which 10 µL of 5 mg/mL ethidium bromide have been added (we use a blunted spinal needle attached to a syringe).