This kit is designed to allow quick and specific assays for eukaryotic type II DNA topoisomerases. This kit facilitates the purification and characterization of type II enzymes and contains all reagents necessary for routine assays of type II topoisomerases. The assay is based upon decatentation of kinetoplast DNA (kDNA) and because it is specific for type II activity (not type I), it can be carried out with crude cell extracts. Reaction products are resolved using a novel gel system developed by TopoGEN that allows extremely rapid and unambiguous detection of topoisomerase II activity. The appropriate buffers, DNA substrates and DNA markers are included. Human Top2a is included with kits denoted with -A in the catalog number. Topoisomerase II decatenated and linear DNA markers are included to allow clear and facile identification of topo II activity in the extracts or fractions defined by the user. The assay kit can also be used in conjunction with purified enzyme (supplied by TopoGEN) to detect inhibitors of topo II. The kDNA assay will detect poisons that stimulate DNA cleavage by topo II as well as agents that simply inhibit catalytic activity. The advantage of kDNA based assays is that the assays are fast and easy to allow activity guided purification of inhibitors from crude mixtures or extracts.
Human Topoisomerase II Assay Kit
Human Topoisomerase II Assay Kit Contents (For 100 assay kit size):
-100 Units Human Top2a Enzyme (available with catalog no. TG1001-1A)
-Kinetoplast DNA (kDNA) 20 ug
-Marker linear kDNA in gel loading buffer
-Marker decatenated kDNA (topo II treated)
-10X Topoisomerase II assay buffer*
-5X Stop buffer/ gel loading dye
-Detailed instruction manual
*To improve stability, the 10x Topo II Assay buffer is provided as two components: Buffer A (no ATP) and Buffer B (ATP). The Complete 10x Assay buffer is made fresh each time by combining Buffer A and B.
Titration of Topo II Activity Using the Human Topoisomerase II Assay Kit
Lane 1: 200ng linearized kDNA
Lane 2: 200ng decatenated kDNA
Lane 3: No Topo II
Lane 4: 1:2 diluted Topo II
Lane 5: 1:4 diluted Topo II
Lane 6: 1:8 diluted Topo II
Lane 7: 1:16 diluted Topo II
Lane 8: 1:32 diluted Topo II
Lane 9: 1:64 diluted Topo II
Human Topoisomerase II Enzyme was incubated with kDNA for 15 min at 37° C using the assay buffer supplied with the kit. The enzyme concentration was approximately 2 units per ul. Reactions were terminated using stop buffer and loaded directly onto a 1% agarose gel containing ethidium bromide (0.5ug/ml). After electrophoresis, the gel was destained for 30 min and photographed. Note that the decatenated products contain open circular (upper band) and covalently closed circular (relaxed) minicircle DNA. Note also that linear DNA migrates between the nicked and relaxed species. The nicked minicircular DNA is present in all kDNA preparations. Additionally, the amount of nicked DNA may vary between KDNA preparation. Linear and decatenated kDNA markers are supplied in the kit. It is important to run these markers to identify positions of different forms. When assaying crude extracts, it is also important to realize that extracts often may contain UV fluorescing contaminants (RNA or DNA breakdown products for example). The markers will help in identifying such artifacts; however, we advise that you also run one lane of protein extract without kDNA substrate to reveal these contaminants.
The most serious complications arise when there are interfering proteins or substances in the extract being assayed. Crude, cell free extracts may contain excessive amounts of DNA binding proteins or positively charged proteins that stick to the DNA and inhibit enzyme access. Also, nuclease contaminants may degrade or nick the kDNA substrate and therefore obscure the results. A good way to deal with this problem includes cleaning up crude extracts by ammonium sulfate precipitation followed by column chromatography. Also, by diluting extracts and/or adding a tRNA carrier (to compete basic proteins), one can sometimes minimize such problems.
This kit is shipped at ambient temperature or on dry ice if purchased with enzyme. Store enzyme at -70° C. The DNAs should be stored at 4° C. The assay buffer components should be stored at -20° C upon receipt.
Reviews and Citations:
- Gardner L, Malik R, Shimizu Y, Mullins N, Elshamy WM: Geminin overexpression prevents the completion of topoisomerase IIα chromosome decatenation, leading to aneuploidy in human mammary epithelial cells. Breast Cancer Research 2011, 13(3): R53. doi: 10.1186/bcr2884
- Bower JJ, Karaca GF, Zhou Y, Simpson DA, Cordeiro-Stone M, Kaufman WK: Topoisomerase IIa maintains genomic stability through decatenation G2 checkpoint signaling. Oncogene 2010, 29(34): 4787-4799. doi: