The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
A unit of topo I can relax 0.2 ug pGEM 5Z DNA in 30 min at 37oC. The enzyme is supplied at a nominal concentration of approximately 2 units/ul.
Quality Control Tests: 1. A test for nuclease contamination is carried out by assaying for the formation of linear KDNA and linear plasmid DNA. Incubation with KDNA or supercoiled pGEM 5Z DNA (4 hrs. at 37ºC in the presence of 10 mM MgCl2) is performed. Linear DNA or breakdown products are not generated under these conditions. 2. A check for cross contamination with Topo II is negative. There must be no decatenation of KDNA in topo II reaction conditions. 3. Performance in converting super-coiled DNA to relaxed DNA.
Possible Applications: Studying the effects of supercoiling on transcription in vitro. Studying chromatin reconstitution in vitro. Determining the degree of supercoiling of naturally occurring DNA. Detecting mutant plasmids that differ in length by only one basepair. Increasing restriction endonuclease digestion of resistant DNA substrates by unwinding the DNA coils to expose restriction sites. Anticancer drug screening. Dilution Buffer: Dilutions should be performed in 1x Reaction Buffer (recipe given below). Assay Conditions: Relaxation assays are carried out in a final volume of 10-20 µl in topo I reaction buffer (40 mM Tris-Cl, pH 7.5, 100 mM NaCl or KCl, 10mM MgCl2, 0.5 mM EDTA, 30 µg/ml BSA). Assays: Supercoiled plasmid DNA is used at 0.2 µg/reaction. Reactions are terminated with 5 µl (per 20 µl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products are analyzed on a 0.8% native agarose gel. Buffer: Supplied with 5X Reaction Buffer [200 mM Tris-HCl (pH 7.5), 500 mM KCl, 50 mM MgCl2, 50 mM DTT, 2.5 mM EDTA and 150 µg/ml BSA].
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Preparation and Storage
Stability and Storage
Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
This product is an active protein and may elicit a biological response in vivo, handle with caution.
DNA topoisomerase 1
DNA topoisomerase I
NUP98 fusion gene
Topoisomerase (DNA) I
Type I DNA topoisomerase
The reaction catalyzed by topoisomerases leads to the conversion of one topological isomer of DNA to another.
Involvement in disease
Note=A chromosomal aberration involving TOP1 is found in a form of therapy-related myelodysplastic syndrome. Translocation t(11;20)(p15;q11) with NUP98.
Belongs to the eukaryotic type I topoisomerase family.
Sumoylated. Lys-117 is the main site of sumoylation. Sumoylation plays a role in partitioning TOP1 between nucleoli and nucleoplasm. Levels are dramatically increased on camptothecin (CPT) treatment.
Nucleus > nucleolus. Nucleus > nucleoplasm. Diffuse nuclear localization with some enrichment in nucleoli. On CPT treatment, cleared from nucleoli into nucleoplasm. Sumolyated forms found in both nucleoplasm and nucleoli.