Active DNA topoisomerase IIalpha is a component of the salt-stable centrosome core.
Recently, we reported that the monoclonal antibody specific for human DNA topoisomerase IIalpha, Ki-S1, stains not only the nuclei of human A431 cells but also extranuclear structures suggestive of centrosomes (Meyer, K. N., Kjeldsen, E., Straub, T., Knudsen, B. K., Kikuchi, A., Hickson, I. D., Kreipe, H., and Boege, F. (1997) J. Cell Biol. 136, 775-788). Here, we confirm colocalization of Ki-S1 with the centrosomal marker gamma-tubulin. In addition, we show labeling of centrosomes by peptide antibodies against the N and C termini of human topoisomerase IIalpha. Probing Western blots of isolated centrosomes with topoisomerase IIalpha antibodies, we demonstrate a protein band of 170 kDa. Moreover, isolated centrosomes exhibited DNA decatenation and relaxation activity correlated to the amount of topoisomerase IIalpha protein in the same way as seen in the pure recombinant enzyme. Topoisomerase IIalpha epitopes could not be removed from centrosomes by salt extraction, DNase treatment, or RNase treatment, procedures that completely removed the enzyme from nuclei. Taken together, these observations suggest that active topoisomerase IIalpha is bound tightly to the centrosome in a DNA-independent manner. Because such centrosomal topoisomerase IIalpha was also present in quiescent lymphocytes devoid of topoisomerase IIalpha in the nuclei, we assume that it might be a long-lived storage form.
Barthelmes
H U
HU
Medizinische Poliklinik der Universität Würzburg, D-97070 Würzburg, Germany.
Grue
P
P
Feineis
S
S
Straub
T
T
Boege
F
F
D000818:Animals; D000951:Antigens, Neoplasm; D002294:Carcinoma, Squamous Cell; D002458:Cell Fractionation; D002478:Cells, Cultured; D018385:Centrosome / Q000201:enzymology*; D016831:Crithidia fasciculata / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000032:analysis* / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000378:metabolism; D004268:DNA-Binding Proteins; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D008214:Lymphocytes / Q000201:enzymology* / Q000648:ultrastructure; D014404:Tubulin / Q000032:analysis; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D018105:DNA, Kinetoplast; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D014404:Tubulin; D004250:DNA Topoisomerases, Type II