The dynamics of DNA topoisomerase IIalpha in living cells

Here, we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino-terminus of human DNA topoisomerase IIalpha (Topo IIalpha) for use in studying the dynamics of Topo IIalpha in living cells. In previous studies, this plasmid was transfected into LLC-Pk cells, a porcine epithelial cell line that remains relatively flat during mitosis. After selection for stable integration, cells were cloned by serial dilution in microwells and used to grow a stable cell line expressing EGFP-Topo IIalpha; this cell line was termed LPk-GT2. Using photobleaching methods with conventional and patterned photobleaching, LPk-GT2 cells were used to demonstrate the rapid dynamics of Topo IIalpha exchange in both interphase nuclei and mitotic chromosomes. These rapid dynamics are dependent on enzyme activity since ICRF159, a catalytic inhibitor of Topo IIalpha, slows dynamics significantly. The methods utilized in these studies are described herein.