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The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress.

Bachant J, Jessen SR, Kavanaugh SE, Fielding CS - J. Cell Biol. (2005)

Bottom Line: Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest.We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication.Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA 92521, USA. jeffbach@citrus.ucr.edu

ABSTRACT
The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore-spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

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Spindle extension in KT mutants during HU or metaphase arrest. cdc23-1 (JBY1289), cdc23-1rad53-21 (JBY1293), cdc23-1ndc10-1 (JBY1367), cdc23-1ndc80-1 (JBY1363), cdc23-1dam1-1 (JBY1333), and cdc23-1ipl1-321 (JBY1357) SPC42-GFP strains were released from G1 ± 200 mM HU at 35°C. After 2.5 h, the distance between Spc42-GFP foci was measured for 250 cells. The percentage of spindles ≥4 μm (−HU) or ≥3 μm (+HU) is depicted.
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fig7: Spindle extension in KT mutants during HU or metaphase arrest. cdc23-1 (JBY1289), cdc23-1rad53-21 (JBY1293), cdc23-1ndc10-1 (JBY1367), cdc23-1ndc80-1 (JBY1363), cdc23-1dam1-1 (JBY1333), and cdc23-1ipl1-321 (JBY1357) SPC42-GFP strains were released from G1 ± 200 mM HU at 35°C. After 2.5 h, the distance between Spc42-GFP foci was measured for 250 cells. The percentage of spindles ≥4 μm (−HU) or ≥3 μm (+HU) is depicted.

Mentions: In the absence of HU, cdc23-1 mutants arrested with 2–4-μm metaphase spindles (average 2.9 ± 0.6 μm), whereas cdc23-1 strains released in the presence of HU displayed the shorter spindles typical of S phase checkpoint arrest (average 2.0 ± 0.5 μm; Fig. 7). Both with and without HU treatment, cdc23-1duo1-2, cdc23-1ctf13-30, and cdc23-1ndc10-2 strains arrested similarly to cdc23-1 controls (unpublished data). Thus, these mutants fall in the anticipated class that do not perturb preanaphase spindle integrity. In contrast, cdc23-1ndc10-1, cdc23-1ndc80-1, and cdc23-1dam1-1 mutants all displayed spindle extension at the cdc23 block. Of these, cdc23-1ndc10-1 and cdc23-1ndc80-1 mutants also displayed untimely extension after HU treatment. A relatively small population (12%) of cdc23-1dam1-1 cells treated with HU did in fact exhibit ≥3-μm spindles. However, there was much greater requirement for Dam1 after completion of S phase, with 92% of cdc23dam1-1 cells exhibiting spindle extension.


The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress.

Bachant J, Jessen SR, Kavanaugh SE, Fielding CS - J. Cell Biol. (2005)

Spindle extension in KT mutants during HU or metaphase arrest. cdc23-1 (JBY1289), cdc23-1rad53-21 (JBY1293), cdc23-1ndc10-1 (JBY1367), cdc23-1ndc80-1 (JBY1363), cdc23-1dam1-1 (JBY1333), and cdc23-1ipl1-321 (JBY1357) SPC42-GFP strains were released from G1 ± 200 mM HU at 35°C. After 2.5 h, the distance between Spc42-GFP foci was measured for 250 cells. The percentage of spindles ≥4 μm (−HU) or ≥3 μm (+HU) is depicted.
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Related In: Results  -  Collection

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fig7: Spindle extension in KT mutants during HU or metaphase arrest. cdc23-1 (JBY1289), cdc23-1rad53-21 (JBY1293), cdc23-1ndc10-1 (JBY1367), cdc23-1ndc80-1 (JBY1363), cdc23-1dam1-1 (JBY1333), and cdc23-1ipl1-321 (JBY1357) SPC42-GFP strains were released from G1 ± 200 mM HU at 35°C. After 2.5 h, the distance between Spc42-GFP foci was measured for 250 cells. The percentage of spindles ≥4 μm (−HU) or ≥3 μm (+HU) is depicted.
Mentions: In the absence of HU, cdc23-1 mutants arrested with 2–4-μm metaphase spindles (average 2.9 ± 0.6 μm), whereas cdc23-1 strains released in the presence of HU displayed the shorter spindles typical of S phase checkpoint arrest (average 2.0 ± 0.5 μm; Fig. 7). Both with and without HU treatment, cdc23-1duo1-2, cdc23-1ctf13-30, and cdc23-1ndc10-2 strains arrested similarly to cdc23-1 controls (unpublished data). Thus, these mutants fall in the anticipated class that do not perturb preanaphase spindle integrity. In contrast, cdc23-1ndc10-1, cdc23-1ndc80-1, and cdc23-1dam1-1 mutants all displayed spindle extension at the cdc23 block. Of these, cdc23-1ndc10-1 and cdc23-1ndc80-1 mutants also displayed untimely extension after HU treatment. A relatively small population (12%) of cdc23-1dam1-1 cells treated with HU did in fact exhibit ≥3-μm spindles. However, there was much greater requirement for Dam1 after completion of S phase, with 92% of cdc23dam1-1 cells exhibiting spindle extension.

Bottom Line: Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest.We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication.Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA 92521, USA. jeffbach@citrus.ucr.edu

ABSTRACT
The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore-spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

Show MeSH
Related in: MedlinePlus