Active DNA topoisomerase II alpha is a component of the salt-stable centrosome core

Recently, we reported that the monoclonal antibody specific for human DNA t opoisomerase II alpha, Ki-S1, stains not only the nuclei of human A431 cell s but also extranuclear structures suggestive of centrosomes (Meyer, K. N., Kjeldsen, E., Straub, T., Knudsen, B. K., Kikuchi, A., Hickson, I. D., Kre ipe, 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 add ition, we show labeling of centrosomes by peptide antibodies against the N and C termini of human topoisomerase II alpha. Probing Western blots of iso lated centrosomes with topoisomerase II alpha antibodies, we demonstrate a protein band of 170 kDa. Moreover, isolated centrosomes exhibited DNA decat enation and relaxation activity correlated to the amount of topoisomerase I I alpha protein in the same way as seen in the pure recombinant enzyme. Top oisomerase II alpha 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 II alpha is bound tightly to the centrosome in a DNA-independent manner. Because such centrosomal topoisomerase II alpha wa s also present in quiescent lymphocytes devoid of topoisomerase II alpha in the nuclei, we assume that it might be a long-lived storage form.