Topo IV

Catalog Number

Description

Size

2000EC-1

E. coli Topo IV

250 units

2000EC-3

E. coli Topo IV

500 units

2000EC-5

E. coli Topo IV

1000 units

2000EC-7

E. coli Topo IV

2000 units

Description:

Topo IV is a type II topoisomerase that is encoded by two genes parC and parE in E. coli (1). Topo IV is essential for decatenation and chromosomal segregation in E. colo; thus, topo IV tends to prefer intermolecular strand passing (catenating and decatenating reactions) over relaxation reactions. Topo IV is also the target for quinolone-based antibacterial agents which act by subverting the enzyme into a DNA damaging agent. TopoGEN offers purified Topo IV for use in all aspects of drug development and screening assays. The enzyme is very active in decatenating KDNA (Cat. # 2031-1) but will also relax supercoiled plasmid DNA (it is less robust in relaxation activity). Topo IV is purified according to Peng and Marians (2).

Quality Control Tests:

Topo IV was overexpressed and purified using the published methods (2). A single band on SDS-PAGE was detected by CB staining for each subunit. Cross contamination by topo I was assessed by assaying for relaxation of supercoiled DNA under conditions optimized for type I activity. Under these conditions, after 2 hours of incubation with pBR233, no relaxation products were detectable.

Assay Conditions:

Reactions contained the following (20 ul): 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 10 mM DDT, 100 mM potassium glutamate, 40 uM ATP, 50 ug BSA/ml, 0.2 ug KDNA. Reactions were terminated after 30 min at 37 C with SDS (1%) followed by proteinase K digestion (50 ug/ml, 15 min at 56 C). Reaction products were run on a 1% agarose gel containing 0.5 ug/ml ethidium bromide (monitored with hand held UV light to resolve decatenation products), destained with distilled water (20 min room temperature) and photographed.

Storage and Shipping Conditions:

The enzyme is shipped on dry ice and should be stored at -70 C. We also recommend that the enzyme be aliquoted after the first thaw. Note that repeated rounds of freeze/thaw will lead to activity loss. The storage buffer is: 1 mM EDTA, 5 mM DTT, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl and 30% glycerol. The enzyme activity is stable for 1-2 days (not longer) on ice.

References

Kato et al. (1990) Cell 63:393-404.
Peng, H., and Marians, K. (1999). DNA Topoisomerase Protocols Volume I; page 163 (available from TopoGEN, Cat. # VI)

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