Rapid exchange of mammalian topoisomerase IIα at kinetochores and chromosome arms in mitosis

A stable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase IIalpha (topollalpha) protein was downregulated and replaced by the expression of topollalpha fused with enhanced green fluorescent protein (EGFP-topollalpha). The EGFP-topolla faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP-topollalpha accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP-topollalpha diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP-topollalpha associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topolla was essential for rapid dynamics, as ICRF-187, an inhibitor of topollalpha, blocked recovery after photobleaching. Although some topollalpha may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topollalpha in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation.