Topoisomerase I

(DNA Relaxing Enzyme)
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Additional Buffer:
(1 ml of each buffer is included with each pack size of enzyme)
 73598 1 ml    $50.00  

Source: Calf thymus

Applications:

  1. Analysis of DNA supercoiling and conformation(2) .
  2. Produces knots in single-stranded, circular DNA(2).
  3. Analysis of chromatin reconstitution in vitro(4).
  4. DNA repair studies(4).
  5. Studies on drug resistance, cell proliferation and leukemia(5,6).

Description:
Topoisomerase I relaxes both positively and negatively supercoiled DNA by a transient single-stranded breakage and rejoining of phosphodiester bonds(1,2). Relaxing supercoiled DNA with Topoisomerase I allows molecules to be differentiated by a single nucleotide pair by gel electrophoresis(3). Topoisomerase I is active in the absence of divalent cations and can be used in solutions containing EDTA. It doesn't require ATP for the activity.

Purity:
Tested for contaminating endonucleases and exonucleases.

Storage Buffer:
20mM potassium phosphate (pH 7.2), 50mM KCl, 0.05mM EDTA, 5mM 2-mercaptoethanol, 0.02% BSA, 50% glycerol.

Assay Conditions: The reaction mixture contains 35mM Tris-HCl, pH 8.0, 72mM KCl, 5mM MgCl2, 5mM DTT, 5mM spermidine, 0.01% BSA, 0.5 µg/µl supercoiled plasmid DNA and enzyme. Incubation is at 37°C for 30 minutes.

Unit Definition:
One unit is the amount of enzyme required to catalyze the conversion of 0.5 µg supercoiled pBR322 DNA to a relaxed state in 30 min at 37°C.

Concentration:
5 units/µl

10X Topoisomerase I Reaction Buffer (1 ml included, PN: 73598): 350mM Tris-HCl, pH 8.0, 720mM KCl, 50mM MgCl2, 50mM DTT, 50mM spermidine. 

BSA (1 ml included, PN 70485): 0.1% BSA in sterilized water. 

Note: This enzyme easily sticks to glass or plastic material. BSA should be added to the reaction to avoid this. Prepare the reaction mixture by adding, in order, Reaction Buffer, nuclease-free water, substrate DNA, BSA (0.01% final concentration) and enzyme. White precipitation may occur when adding 0.1% BSA directly to the Reaction Buffer.

Analyze relaxed DNA on an agarose gel. Topoisomerase I treated relaxed DNA resumes its supercoiled configuration if treated with EtBr. Therefore, electrophoresis should be done with an agarose gel that does not contain EtBr. Stain with EtBr after running the gel.

References:

  1. GELLERT, M. (1981) Annu. Rev. Biochem. 50, 879-910.
  2. WANG, J. C. (1979) Proc. Natl. Acad. Sci. USA 76, 200-203.
  3. LUCKOW, B., RENKAWITZ, R. AND SCHUTZ, G. (1987) Nucl. Acids Res. 15,
    417-429.
  4. OSHEROFF, N. (1989) Pharmacol. Ther. 41, 223-241.
  5. LIU, L. F. (1989) Annu. Rev. Biochem. 58, 351-357.
  6. BODLEY, A. L., LIU, L. F. (1988) BioTechnology, 6, 1315-1319.

Storage:
Shipped on dry ice. Store at -20°C.

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