Unique functions of mammalian DNA-topoisomerases II␣ and -␤ are suggested by their distinct cellular distribution and chromatin binding at mitosis. Here, we studied H69-VP cells that, due to a homozygous mutation, express topoisomerase II␣ mostly outside the nucleus. In these cells topoisomerase II␤ showed a normal nuclear localization. However, at mitosis it diffused away from the chromatin despite the nuclear lack of the ␣-isoform. 80% of these cells performed chromosome condensation and disjunction with the aid of cytosolic topoisomerase II␣, which bound to the mitotic chromatin with low affinity. However, the genotype of these cells was highly polyploid indicating an increased rate of non-disjunction. In 20% of the mutant cells neither topoisomerase II isoform was bound to the mitotic chromatin, which appeared as an unstructured DNA spheroid unable to undergo disjunction and cytokinesis. Parental H69 cells expressing topoisomerase II␣ inside the nucleus exhibited high affinity binding of the enzyme to the mitotic chromatin. Their genotype was mostly diploid and stable. We conclude (i) that high affinity chromatin binding of topoisomerase II␣ is essential for chromosome condensation/disjunction and (ii) that topoisomerase II␤ does not adopt these functions.

DNA-topoisomerase I has been implied in RNA splicing because it catalyzes RNA strand transfer and activates serine/arginine-rich RNA-splicing factors by phosphorylation. Here, we demonstrate a direct interaction between topoisomerase I and pyrimidine tract binding protein-associated splicing factor (PSF), a cofactor of RNA splicing, which forms heterodimers with its smaller homolog, the nuclear RNA-binding protein of 54 kDa (p54 nrb ). Topoisomerase I, PSF, and p54 nrb copurified in a 1:1:1 ratio from human A431 cell nuclear extracts. Specific binding of topoisomerase I to PSF (but not p54 nrb ) was demonstrated by coimmunoprecipitation and by far Western blotting, in which renatured blots were probed with biotinylated topoisomerase I. Chemical cross-linking of pure topoisomerase I revealed monomeric, dimeric, and trimeric enzyme forms, whereas in the presence of PSF/p54 nrb the enzyme was cross-linked into complexes larger than homotrimers. When topoisomerase I was complexed with PSF/p54 nrb it was 16-fold more active than the pure enzyme, which could be stimulated 5-and 16-fold by the addition of recombinant PSF or native PSF/p54 nrb , respectively. A physiological role of this stimulatory mechanism seems feasible, because topoisomerase I and PSF showed a patched colocalization in A431 cell nuclei, which varied with cell cycle.

Recently, we reported that the monoclonal antibody specific for human DNA topoisomerase II␣, 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 ␥-tubulin. In addition, we show labeling of centrosomes by peptide antibodies against the N and C termini of human topoisomerase II␣. Probing Western blots of isolated centrosomes with topoisomerase II␣ antibodies, we demonstrate a protein band of 170 kDa. Moreover, isolated centrosomes exhibited DNA decatenation and relaxation activity correlated to the amount of topoisomerase II␣ protein in the same way as seen in the pure recombinant enzyme. Topoisomerase II␣ 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␣ is bound tightly to the centrosome in a DNA-independent manner. Because such centrosomal topoisomerase II␣ was also present in quiescent lymphocytes devoid of topoisomerase II␣ in the nuclei, we assume that it might be a long-lived storage form.We have observed (1, 2) that the monoclonal antibody Ki-S1 directed against DNA topoisomerase II␣ (3) labels not only the cell nuclei (a long-standing observation) but also labels small globular structures located at the poles of mitotic spindles (a new finding). Ki-S1 labeling of extranuclear globules was reproducible in two human cell lines, whereas a similar observation was not made with antibodies directed against DNA topoisomerase I or II␤. We assumed that these Ki-S1-positive extranuclear structures would most likely be centrosomes. However, they could just as well be chromosome fragments or micronuclei, or the staining could be due to a spurious crossreaction of the Ki-S1 antibody with some centrosomal protein other than topoisomerase II␣. However, if such artifacts were excluded, our observation might indicate topoisomerase II␣ as a possible component of centrosomes. We found our chance observation interesting enough to be followed up. Here, we carried out immunocytochemical colocalization studies combining antibodies against ␥-tubulin (an established centrosomal marker) with antibodies against several distinct epitopes of DNA topoisomerase II␣. We corroborated such studies by biochemical analysis of isolated centrosomes. Our results confirm that active DNA topoisomerase II␣ is associated with centrosomes.

No abstract