Reagents
VACCINIA DNA TOPOISOMERASE I |
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| Product Information: VACCINIA DNA TOPOISOMERASE I from Vaccinia virus is a type IB topoisomerase. Among other topoisomerases it is distinguished by its small size (314 amino acids) and the site-specificity in DNA cleavage-religation. In DNA duplexes the enzyme binds to a characteristic pentapyrimidine motif 5' (C/T)CCTT↓ and cleaves it at the 3' side. The T↓ nucleotide remains covalently linked to Tyr274 of the topoisomerase, which subsequently religates the strand back. This permits relaxation of supercoiled double stranded DNA by removing both right- and left-handed supercoils from covalently closed DNA duplexes. Under special conditions the enzyme can join the cleaved DNA with pentapyrimidine motif to another DNA duplex and can therefore create recombinant DNA molecules. VACCINIA DNA TOPOISOMERASE I is a well-researched enzyme and is the select member of topoisomerase IB family for which the most detailed kinetic data is available. The enzyme is used in many fields of research in molecular and cell biology and in experimental pathology. 1 pmole of the enzyme equals approximately 500 Units. |
Applications: - Detection of apoptosis in tissue sections and in fixed cells In situ analysis of different types of apoptotic DNA degradation. - As a molecular engine in Nanobiotechnology In FRET probes and techniques In vitro transcription studies. - Studies of chromatin reconstitution Topoisomerase I mediated TA cloning Studies of DNA supercoiling. - Detection of plasmid mutations accompanied by the minimal increase of their length (a single nucleotide sensitivity). - Optimizing and enhancing DNA digestion by restrictases and DNases. |
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| Unit Definition: One unit of Vaccinia DNA Topoisomerase I is defined as the amount of enzyme that catalyzes conversion of 1 µg of supercoiled DNA, Form I (pUC19 RF I (negatively supercoiled) to relaxed closed circular DNA (Form II) in 1 hour at 37°C under standard assay conditions. DNA supercoiling is assessed by agarose gel electrophoresis. Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT. |
References: 1. Shuman S, Golder M, Moss B. J Biol Chem. 1988 Nov 5;263(31):16401-7. PMID: 2846543 (free article). 2. Shuman, S. (1992) J. Biol. Chem. J Biol Chem. 1992 Apr 25;267(12):8620-7. PMID: 1314832. (free article). 3. Sekiguchi J, Cheng C, Shuman S. J Biol Chem. 1997 Jun 20;272(25):15721-8. PMID: 9188465 (free article). 4. Minchew CL, Didenko VV. Molecules. 2011 Jun 3;16(6):4599-614. PMID: 21642935. |
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Storage temperature: -20ºC. Quality Assurance Statement: Purified free of contaminating exonucleases and endonucleases. |
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