Kinetoplast DNA Decatenation Assay with Eukaryotic Topoisomerase II.

The interlinking of DNA during replication and other DNA processes is resolved by type II DNA topoisomerases. In humans, topoisomerase IIα is the main enzyme involved in this process using a transient double-stranded DNA break to unlink sister chromatids during and after replication to allow for mitosis. To measure decatenation, a consistent catenated substrate called kinetoplast DNA (kDNA) is commonly used. kDNA is often purified from the trypanosome Crithidia fasciculata. kDNA represents catenated or interlinked circles of DNA. Early work on topoisomerases identified kDNA as a useful substrate for measuring decatenation by DNA topoisomerase II. This protocol will describe a common method for measuring decatenation of kDNA by eukaryotic type II topoisomerases.