DNA Topoisomerase I, Vaccinia

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Last modified: 2008-07-15 14:57:15

Topoisomerase I from vaccinia virus is a type I eukaryotic topoisomerase that removes both positive and negative superhelical turns (also called right- and left-handed supercoils) from covalently closed DNA. The product of the reaction is a covalently closed, circular DNA with fewer positive or negative superhelical turns. demonstrates the activity of Topoisomerase I on supercoiled plasmid DNA.
DNA Topoisomerase I does not absolutely require Mg2+ to function, although low concentrations of magnesium ion may increase activity. The protein has a molecular weight of 32,000 daltons.

Figure 1. Relaxation of 1µg of supercoiled pUC19 by Vaccinia Topoisomerase I. Plasmid DNA treated with Topoisomerase I was analyzed by agarose gel electrophoresis in a 1% agarose gel stained with ethidium bromide. Lane 1 contains the starting DNA; the plasmid shows progressive relaxation from left to right as the enzyme concentration increases. Lane M, DNA ladder

Unit Definition:

One unit will convert 1µg of supercoiled DNA (Form I) to relaxed closed circular DNA (Form II) in 1 hour at 37°C in 40mM Tris-acetate, pH 8.3, 0.1mM EDTA, 2.5mM MgCl₂, and 100mM NaCl.

Storage Buffer:

50% glycerol solution containing 50mM Tris-HCl, pH 7.5, 0.1M NaCl, 0.1mM EDTA, 1mM DTT, and 0.1% Triton® X-100.

Quality Control:

Vaccinia Topoisomerase I is tested with supercoiled DNA and is free of detectable exo- and endonuclease, and RNase activities.

Last modified: 2008-05-14 16:46:46

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 base-pair
• Increasing restriction endonuclease digestion of resistant DNA substrates by 'unwinding' the DNA coils to expose restriction sites

Last modified: 2008-05-14 16:46:46

Protocols for: DNA Topoisomerase I, vaccinia

(catalogue number VT710500 / VT7101K / VT7105K)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46