E. coli Topoisomerase IV (top4) is a type II topoisomerase that is encoded by two genes parC and parE in E. coli. This enzyme is essential for decatenation and chromosomal segregation in E. coli; thus, topo IV tends to prefer intermolecular strand passing (catenating and decatenating reactions) over relaxation reactions. EC Topoisomerase IV is also the target for quinolone-based antibacterial agents which act by subverting the enzyme into a DNA damaging agent. TopoGEN offers purified Top4 for use in all aspects of drug development and screening assays. The enzyme is very active in decatenating kinetoplast DNA (kDNA) but will also relax supercoiled plasmid DNA. It is less robust in relaxation activity, however. E. coli Topoisomerase IV is purified according to Peng and Marians.
E. coli Topoisomerase IV
A 2-part assay buffer and dilution buffer are included.
Top4 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.
Quality Control Tests:
Top4 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 pBR322, no relaxation products were detectable.
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.
Reviews and Citations:
- Phillips JW, Goetz MA, Smith SK, Zink DL, Polishook J, Onishi R, Salowe S, Wiltsie J, Allocco J, Sigmund J, Dorso K, Lee S, Skwish S, de la Cruz M, Martin J, Vicente F, Genilloud O, Lu J, Painter RE, Young K, Overbye K, Donald RGK, Singh SB: Discovery of Kibdelomycin, A Potent New Class of Bacterial Type II Topoisomerase Inhibitor by Chemical-Genetic Profiling in Staphylococcus aureus. Cell Chemistry and Biology 2011, 18: 955-965. doi: 10.1016/j.chembiol.2011.06.011.
- Peng, H., and Marians, K. (1999). DNA Topoisomerase Protocols Volume I; page 163.