kDNA is the ideal substrate for topoisomerase II assays because it is specific for type II reaction mechanisms. Researchers can even assay for a type II enzyme in the presence of large excess of topoisomerase I. Thus, kDNA works well to quantify type II activity in crude cell extracts, which are frequently overloaded with topoisomerase I. The kDNA substrate works exceptionally well with human Top 2a and b as well as prokaryotic enzymes, such as DNA gyrase or topoisomerase IV. This substrate is packaged with our Topo II Assay Kit, Topo II Drug Kit, and Gyrase Assay Kits.
Kinetoplast DNA (kDNA)
Assays employing crude extracts for topo II activity based upon relaxation of supercoiled DNA can be complicated due to the presence of topo I in partially purified fractions. Additional complications arise with contaminating nuclease activity (due to Mg++) which degrade or nick the supercoiled substrate. These problems can be avoided by using a catenated DNA substrate prepared from the kinetoplast of the insect trypanosome Crithidia fasciculata. Kinetoplast DNA (kDNA) is an aggregate of interlocked DNA minicircles (mostly 2.5 kb) that form extremely large networks of high molecular weight. As a result, these networks fail to enter an agarose gel. Upon incubation with topo II, which engages DNA in a double stranded breaking and reunion cycle, minicircular DNAs are effectively released (decatenated). The decatenated minicircles move rapidly into the gel owing to their small size. This reaction will not occur with topoisomerase I. The products of the reaction can vary as follows:
Quality Control Tests:
- For catenated KDNA substrate, at least 90% of the DNA will be retained in the well of a 1% agarose gel.
- Decatenation of each batch of Kinetoplast DNA is tested with purified topoisomerase II.
kDNA is stored in 10 mM Tris-Cl (pH 7.5), and 1 mM EDTA at the concentration specified with the product.
kDNA should be stored at 4°C; however, long term storage at -20°C is acceptable. The DNA is usually shipped at ambient temperature.
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.