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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00977

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Functional compatibility between isoform and ß of type II DNA topoisomerase

Ayako Sakaguchi^* and Akihiko Kikuchi

Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School and Faculty of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

View larger version (53K): [in a new window]   Fig. 1. Knockdown of each topo II isoform by siRNA. (A,B) HeLa cells were transfected with siRNA-1 (A1), siRNA-2 (A2), ß siRNA-1 (B1) and ß siRNA-2 (B2) or buffer as a control (A and B; C). Cells were assayed on the second, third and fourth days after transfection by immunoblotting using the anti-topo II antibody 8D2 (left panels) and the anti-topo IIß antibody 3B6 (right panels). (C) Gene silencing was also confirmed by immunofluorescence using the anti-topo II antibody 8D2 (for siRNA-1-knockdown cells, upper panels) or the anti-topo IIß antibody 1A5 (for ß siRNA-2-knockdown cells, lower panels); cells were also stained with DAPI. Ab: antibody. Bar, 20 µm.

View larger version (30K): [in a new window]   Fig. 2. Cell-cycle analysis using the LSC2. (A) Using LSC2, cells can be sorted into cell-cycle stages (Kawasaki et al., 1997). Cells in red boxes are at prophase and metaphase, and those in blue boxes are at anaphase and telophase. (B) Topo II-knockdown, topo IIß-knockdown and mock-transfected cells were sorted using the LSC2 3 days after transfection (plots). The tables show the number of cells in each category; red boxes correspond to prophase/metaphase cells, blue boxes to anaphase/telophase cells, 2C to G2 phase cells and `total mitotic' is the sum of prophase/metaphase and anaphase/telophase cells. Each transfection was repeated independently at least three times and samples were prepared in four separate Akura films for every experiment.

View larger version (77K): [in a new window]   Fig. 3. Chromosome segregation occurs in topo II-knockdown cells. (A,a-c) Three representatives in mitotic stages were depicted from mock-transfected cells. (B,a-f) Three days after transfection, topo II-knockdown cells were stained with DAPI, anti-topo II antibody (8D2) and anti-tubulin antibody (YL1/2). (C,a,b) Threads of DNA are indicated by arrowheads. (D) Topo II-knockdown cells were also stained with anti-NPC (nuclear pore complex protein) antibody. (E) The number of nuclei connected by a thread of DNA was counted. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 4. Knockdown of both topo II isoforms by siRNAs. (A) HeLa cells were transfected with both siRNA-1 and ß siRNA-2 (1; 5 µl each siRNA was added per 35 mm dish; 2; 10 µl each) or buffer as a control. Cells were assayed on the third or fourth day after transfection by immunoblotting using the anti-topo II antibody 8D2 (left) and the anti-topo IIß antibody 3B6 (right). (B) The topo IIß-knockdown cells were analyzed using the LSC2 3 days after transfection. (C) Topo IIß-knockdown cells (3 days after transfection) were stained with DAPI, anti-topo IIß antibody (6H8) and anti-tubulin antibody (YL1/2). Bar in C, 20 µm (upper panels) and 10 µm (lower panels).

View larger version (40K): [in a new window]   Fig. 5. Topo II activity in knockdown cells. The enzymatic activity of topo II was analyzed by a decatenation assay using kinetoplast DNA as a substrate. We inoculated 4x105 cells and prepared extracts for topo II assays from control (A), topo II-knockdown (B) and topo IIß-knockdown (C) cells 3 days after transfection. In each reaction mixture, a serial twofold dilution of topo II extracts was added. Lane 1: 1/1280 dilution; lane 2: 1/640; lane 3: 1/320; lane 4: 1/160; lane 5: 1/80; lane 6: 1/40; lane 7: 1/20; lane 8: 1/10; lane 9: 1/5; lane 10: undiluted extract. C1: no topo II extract; C2: undiluted extract without ATP; C3: 1/5 dilution without ATP; M1: linear kinetoplast DNA marker (**); M2: decatenated kinetoplast DNA marker (*). (D) Crude extracts immunoblotted using the anti-topo IIß antibody 6H8.

View larger version (55K): [in a new window]   Fig. 6. Chromosome condensation and chromosome axis shortening. (A) Metaphase chromosomes of control (a,b), topo II-knockdown (c,d) and topo IIß-knockdown (e,f) cells were spread and stained with DAPI (a,c,e) and the anti-topo IIß antibody 7B9 (b,f) or the anti-topo II antibody 8D2 (d). In control cells, chromosome axes and centromeres were stained with anti-topo IIß antibody. Bar, 10 µm. (B) Frequency distribution of the length of metaphase chromosomes in control cells and topo IIß-knockdown cells.

View larger version (92K): [in a new window]   Fig. 7. Localization of topo IIß in mitotic chromosomes of topo II-knockdown cells. Mitotic chromosomes of both control (a,b) and topo II-knockdown (c,d) cells on the third day after transfection were stained with anti-topo IIß antibody (3B6) and DAPI. Bar, 10 µm.