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.
Daum
John R
JR
Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
Mo
Yin Yuan
YY
Gorbsky
Gary J
GJ
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002453:Cell Cycle; D002460:Cell Line; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D011929:Razoxane / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D059005:Topoisomerase II Inhibitors; D011929:Razoxane; D004250:DNA Topoisomerases, Type II