Abstract
Here, we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino-terminus of human DNA topoisomerase IIα (Topo IIα) for use in studying the dynamics of Topo IIα 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 IIα; 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 IIα exchange in both interphase nuclei and mitotic chromosomes. These rapid dynamics are dependent on enzyme activity since ICRF159, a catalytic inhibitor of Topo IIα, slows dynamics significantly. The methods utilized in these studies are described herein.
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Acknowledgments
Support was provided by the National Institutes of General Medical Sciences, from the Oklahoma Medical Research Foundation and from the McCasland Foundation. We thank Dr. Penny Tavormina for helpful suggestions.
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Daum, J.R., Mo, Y.Y., Gorbsky, G.J. (2009). The Dynamics of DNA Topoisomerase IIα in Living Cells. In: Clarke, D. (eds) DNA Topoisomerases. Methods in Molecular Biology™, vol 582. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-340-4_18
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DOI: https://doi.org/10.1007/978-1-60761-340-4_18
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