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First published online 31 August 2004
doi: 10.1242/jcs.01351

Journal of Cell Science 117, 4769-4778 (2004)
Published by The Company of Biologists 2004
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Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity

Thein Z. Win1, Adele Goodwin2, Ian D. Hickson2, Chris J. Norbury3 and Shao-Win Wang1,*

1 Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
2 Cancer Research UK Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK
3 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK



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  		Fig. 1. top3-134 mutants accumulate defects during S phase. Wild type (top3+) and top3-134 cells were arrested in G1 by nitrogen starvation and released into nitrogen-rich medium to restart the cell cycle at the non-permissive temperature of 36°C. Cells harvested at hourly intervals were processed for flow cytometry (A), and mitotic index was assessed by scoring bi-nucleate cells (B). The percentage of cells displaying aberrant mitosis with chromosomes lagging between the separating daughter nuclei (indicated by arrowheads in D) was also determined (C). (D) Fluorescence micrographs of DAPI-stained wild type and top3-134 cells, 6 hours after release from nitrogen starvation at 36°C. Bar, 10 µm.

 


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  		Fig. 2. Chromosome segregation defects in top3-134 cells require passage through S-phase. G1-arrested wild type (top3+) and top3-134 cells were released into nitrogen-rich medium to restart the cell cycle at the permissive temperature of 26°C for 5 hours before shifting to the restrictive temperature of 36°C. Cells harvested at hourly intervals were processed for flow cytometry (A), and assessed for mitotic index (B) and aberrant mitosis (C) as in Fig. 1.

 


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  		Fig. 3. The DNA damage checkpoint is activated in top3 mutants. (A) Fluorescence micrographs of DAPI-stained top3-134, top3-134 {Delta}chk1, top3-134 {Delta}cds1 and top3-134 {Delta}rad1 cells grown at the permissive temperature of 26°C or after shift to 36°C for 9 hours. Bar, 10 µm. (B) At 3-hour intervals, samples from cultures of wild type (972), top3-134 and {Delta}rad1 top3-134 cells incubated at the restrictive temperature of 36°C were taken to score the percentage of aberrant mitosis in each culture. (C). One thousand cells from B at the times indicated were plated onto YES plates to assess cell survival. Colonies were counted after 5 days growth at 26°C.

 


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  		Fig. 4. Depletion of Top3 leads to phosphorylation of Chk1. (A) Growth curves of exponentially growing cultures of the top3-P41 strain in the presence ({circ}) or absence ({square}) of thiamine at 36°C. (B) Cell and nuclear morphologies of samples from A were determined by fluorescence microscopy of DAPI-stained cells 12 hours after the addition of thiamine. Bar, 10 µm. (C) Whole-cell protein extracts were prepared by trichloroacetic acid precipitation following glass bead disruption. The extracts were separated by SDS-PAGE and subjected to immunoblotting using anti-HA (upper panel) or anti-Cdc2 (loading control) antibodies as indicated.

 


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  		Fig. 5. top3-134 is synthetically lethal in combination with deletion of rad3/rad26 but not with deletion of other checkpoint genes. (A-D) Tetrads derived from diploid strains of the following genotypes were microdissected onto YES agar: (A) h+/h- rad3::ura4+/rad3+ top3-134/top3+; (B) h+/h- rad26::ura4+/rad26+ top3-134/top3+; (C) h+/h- rad1::ura4+/rad1+ top3-134/top3+; and (D) h+/h- rad17::ura4+/rad17+ top3-134/top3+. Colonies resulting from nine tetrads in each case were photographed after seven days growth at 26°C. The genotypes of the segregants were determined by replica plating and are indicated schematically [right: (A) W, top3+ rad1+ rad3+ rad17+ rad26+; T, top3-134, R, {Delta}rad3; (B) {Delta}rad26; (C) {Delta}rad1; or (D) {Delta}rad17; (A) RT, top3-134 {Delta}rad3; (B) top3-134 {Delta}rad26; (C) top3-134 {Delta}rad1; (D) or top3-134 {Delta}rad17]. Boxes (left) indicate the position of top3-134 double mutants with rad1, rad3, rad17 and rad26, respectively.

 


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  		Fig. 6. Pulsed-field gel electrophoresis analysis of chromosomes from the top3-134 mutant. (A-C) Equal numbers of cells were prepared in agarose gel plugs from exponentially growing cultures of wild type (top3+) or top3-134 cells (four independent isolates) at the permissive temperature of 26°C (A), after nitrogen starvation to arrest cells in G1 (B), or following HU block (3 hours at 36°C) and release (C). Cells were harvested at the time of HU addition (C) and at hourly intervals after removal of HU for up to 3 hours. Arrowheads indicate the aberrant chromosome III band seen in top3-134 samples. (D) Flow cytometric analysis of the DNA content of ethanol-fixed, Sytox Green-stained samples from the experiment shown in C. (E) Samples from C taken at the times indicated were used to score the percentages of aberrant mitosis in each culture.

 


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  		Fig. 7. Aberrant nucleolar structures in the top3-134 mutant. (A) Living gar2-GFP (top3+) and gar2-GFP top3-134 (top3-134) cells were observed by green fluorescence microscopy. Arrowheads indicate cells with aberrant nucleolar structures. (B) Merged images (lower panels) of fluorescence micrographs showing Gar2-GFP (upper panels) and DNA (Hoechst 33342, middle panels) localisation in living cells. (C) Visualisation of lagging Gar2-GFP signal in top3-134 cells. Individual gar2-GFP and gar2-GFP top3-134 cells were observed as in A, over a 20 minute period, with images collected every 4 minutes. Bar, 10 µm.

 

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© The Company of Biologists Ltd 2004