Purification and characterization of topoisomerase IIA from Arabidopsis thaliana

Topoisomerase II (Topo IIA) is an essential ubiquitous enzyme involved in controlling DNA topology during multiple processes of DNA metabolism. Although topoisomerase II from bacteria, viruses, and animals has been extensively characterized, only a few studies have characterized plant topoisomerase II from either crude or partially purified organellar and nuclear extracts, or produced by heterologous expression in Escherichia coli. The aim of this study was to develop a rapid, simple protocol for purification of nuclear topoisomerase II from Arabidopsis thaliana seedlings for kinetic characterization. We developed a reproducible five-step protocol that yielded large quantities of active, stable topoisomerase II purified ca. 1100-fold with 18% yield. Mass spectroscopy analysis of the purified enzyme identified locus At3g23890 encoding the only topoisomerase IIA in the Arabidopsis nuclear genome, providing the first direct functional annotation of this gene. Kinetic properties, substrate specificity, and optimal reaction conditions were determined for the purified Arabidopsis Topo IIA. Arabidopsis Topo IIA showed greater lability at higher temperatures and pH and low activity in the presence of divalent ions other than Mg2+ and exhibited a processive mode of relaxing supercoiled DNA in contrast to the mammalian, insect, and microbial enzymes. This simple and reliable procedure should be useful in purification of Topo IIA from other plant sources.