Topoisomerase II, Alpha
(DNA Relaxing Enzyme)
Alters the topological state of nucleic acids by passing an intact DNA helix through a transient break which generates a separate DNA helix
Source:
E. coli containing a clone of the human Topoisomerase II gene.
Description:
Topoisomerase II alters the topological state of nucleic acids by passing an intact DNA helix through a transient break which generates a separate DNA helix(1,2). As a result of its double-stranded DNA passage mechanism, the enzyme can relax negatively or positively supercoiled DNA, as well as catenate/decatenate or knot/unknot DNA molecules. Topoisomerase II has an absolute requirement for divalent cation and ATP (or dATP).
Properties:
Molecular Weight: 340 kDa homodimer
Optimum Temperature: 30°–37°C
Optimum pH: 7.9
Requirements for Divalent Cation: 5mM Mg2+ is optimum for catalytic activity and 5mM Ca2+
for DNA cleavage.
Optimum ATP Concentration: 0.5 to 1.0mM
Optimum Ion Strength: 100-170mM KCl/NaCl
Inhibitors: N-ethylmaleimide, zinc, novobicin, coumermycin A1, etoposide, amsacrine
Purity:
Greater than 98% pure as determined by silver-stained SDS-PAGE. Free of contaminating exonucleases and endonucleases.
Storage Buffer:
15mM sodium phosphate (pH 7.1), 700mM NaCl, 0.1mM EDTA, 0.5mM DTT, 50% glycerol.
Assay Conditions:
The reaction mixture contains 10mM Tris-HCl, pH 7.9, 175mM KCl, 0.1mM EDTA, 5mM MgCl2, 2.5% glycerol, 1mM ATP.
Unit Definition:
One unit is the amount of enzyme required to fully relax 0.3 μg (5nM) of negatively supercoiled pBR322 plasmid DNA in 15 minutes at 30°C under the standard assay conditions.
Concentration:
20 units/μl
Tested User Friendly™ Functional Test:
Tested for activity in standard Topoisomerase II unit assay.
Functionally Tested 10X Topoisomerse II Reaction Buffer (1 ml included, PN 73592):
100mM Tris-HCl (pH 7.9), 500mM NaCl, 500mM KCl, 50mM MgCl2, 1mM EDTA, 150 μg/ml BSA, 10mM ATP.
Topoisomerase II Dilution Buffer (1 ml included, PN 73591):
10mM sodium phosphate (pH 7.1), 50mM NaCl, 0.2mM DTT, 0.1mM EDTA, 0.5 mg/ml BSA, 10% glycerol.
References:
- BJERGBAEK, L., KINGMA, P., NIELSEN, I. S., WANG, Y., WESTERGAARD, O., OSHEROFF, N. AND ANDERSON, A. H. (2000) J. Biol. Chem. 275, 13041-13048.
- BROMBERG, K. D., HENDRICKS, C., BURGIN, A. B. AND OSHEROFF, N. (2002)
J. Biol. Chem. 277, 31201-31206. - RENODON-CORNIERE, A., JENSEN, L. H., NITISS, J. L., JENSEN, P. B. AND SEHESTED, M. (2002) Biochemistry 41, 13395-13402.
- SABOURIN, M. AND OSHEROFF, N. (2000) Nucl. Acids. Res. 28, 1947-1954.
Applications:
- Relaxing negatively or positively supercoiled DNA.
- Catenating or decatenating DNA.
- Knotting or unknotting DNA.
Storage:
Shipped on dry ice. Store at -20°C for frequent use. Store at -80°C for long-term storage.