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.

Plasmid DNA Cleavage Assay with Eukaryotic Topoisomerase II.

Measuring DNA cleavage levels has been a long-standing method in the field of topoisomerases. Due to the formation of a reversible covalent enzyme:DNA linkage, topoisomerase II activity can be monitored under varying conditions by means of measuring single- and double-stranded DNA breaks. This chapter will provide a method for measuring plasmid DNA cleavage levels generated by eukaryotic topoisomerase II with a specific focus on human type IIA topoisomerases.

Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity.

Human topoisomerase IIα and IIβ regulate DNA topology and knots in chromosomes during crucial cellular processes, making these enzymes common targets for anticancer drugs. However, selective inhibition of topoisomerase IIα (TOP2A) is desired to decrease adverse effects, which may be mediated by topoisomerase IIβ (TOP2B). The main region of difference between the two isoforms is the intrinsically disordered C-terminal domain (CTD), which is being studied as a target for selective inhibition.