Description:
DNA topoisomerase IIa alters the topological state of nucleic acids by passing an intact DNA helix through a transient break which generates a separate DNA helix by changing linking number by 2 for each reaction. As a result of its double-stranded DNA passage mechanism, Topo IIa can relax negatively or positively supercoiled DNA, as well as catenate/decatenate or knot/unknot DNA molecules. Topo IIa has an absolute requirement for divalent cation and ATP. LAE human topoisomerase IIa is produced from a baculovirus expression system, purified from freshly extracted cell lysates, and purified to homogeneity using methods developed by LAE Biotechnology Co., LTD. The final fraction of enzyme contains a single polypeptide band of 170 kDa and is devoid of contaminating nuclease activity.

Applications:
l. Studying the effects of supercoiling on transcription in vitro.
2. Studying chromatin reconstitution in vitro.
3. Determining the degree of supercoiling of naturally occurring DNA.
4. Anticancer drug screening.

Unit Definition:
A unit of topo IIa can relax 0.2 ug pGEM 5Z DNA in 30 min at 37^oC. 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 37^oC 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 I is negative. There must be no relaxation of supercoiling plasmid DNA in the reaction conditions without ATP.

Storage Buffer:
The final fraction of topoisomerase IIa is an affinity column pool and is in the following buffer: 10 mM Tris-Cl, pH 7.5, 500 mM NaCl, 500 mM Imidazol, 0.5 mM PMSF, 1 mM DTT, 10 % glycerol. The enzyme is stable in this buffer at -70^oC. In general the enzyme is less stable when diluted. We recommend that you do not dilute the enzyme before storage at -70^oC. In addition, it is a good practice to aliquot the enzyme after the first thaw.

Dilution Buffer:
Dilutions should be performed in 1x Reaction Buffer (recipe given below).

Buffer:
5X Reaction Buffer (cat# B003) [200 mM Tris-HCl (pH 7.5), 500 mM KCl, 50 mM MgCl₂, 50 mM DTT, 2.5 mM EDTA and 150 ug/ml BSA].

Quality Control:
This product has passed the following quality control assays: absence of detectable endodeoxyribonuclease, exodeoxyribonuclease, and phosphatase activities; performance in converting super-coiled DNA to relaxed DNA.

Legal consideration: FOR RESEARCH USE ONLY.

Shipping/Storage:
The enzyme is shipped on ice and must be stored at -20 degree or lower.

Reference:
Wang, J.C. (1996) Annu. Rev. Biochem. 65: 635-692.