E.coli-Topoisomerase-IV-Enzyme-Data-Sheet

Topoisomerase IV 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.

Topoisomerase IV 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.

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.