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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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Related in: MedlinePlus

Spindle dynamics in rad53-21 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP strains were released from G1 into 200 mM HU. Cells were prepared for imaging 75–90 min after release. Stacks of Spc42-GFP images were acquired every 2 min. Numbers indicate lapsed time. (A) 20-min time-lapse sequence for a WT cell. (B and C) SPB extension and collapse in two HU-treated rad53-21 cells. (D) Graphs depicting changes in spindle length over time for four WT and three rad53-21 cells.
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fig2: Spindle dynamics in rad53-21 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP strains were released from G1 into 200 mM HU. Cells were prepared for imaging 75–90 min after release. Stacks of Spc42-GFP images were acquired every 2 min. Numbers indicate lapsed time. (A) 20-min time-lapse sequence for a WT cell. (B and C) SPB extension and collapse in two HU-treated rad53-21 cells. (D) Graphs depicting changes in spindle length over time for four WT and three rad53-21 cells.

Mentions: We continued our analysis of the rad53 S phase checkpoint defect by visualizing spindle extension using live cell imaging (Videos 1–5, available at http://www.jcb.org/cgi/content/full/jcb.200412076/DC1). WT and rad53-21 SPC42-GFP strains were released from G1 arrest into 200 mM HU media. Filming was initiated once spindle extension was occurring in ∼25% of the population, allowing visualization of the initial extension of the spindle in some cells and subsequent changes once spindle extension had occurred in others. In HU-arrested WT cells, spindle length remained relatively constant at ∼2 μm, although translocations of the spindle within the cell were observed (Fig. 2 A). Spindle translocations were also observed in rad53 mutants, but were accompanied by SPB separation at rates comparable to the 0.5 μm/min observed during the rapid phase of anaphase B (Straight et al., 1997; Fig. 2, B–D). Unexpectedly, spindle extension was in fact reversible, with SPBs contracting at rates similar to the rate of extension. This cycle was variable, with seemingly stochastic conversions between extension and collapse. These dynamics account for the continuum of spindle lengths in HU-treated rad53 cells and are distinct from unidirectional spindle elongation during anaphase.


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 dynamics in rad53-21 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP strains were released from G1 into 200 mM HU. Cells were prepared for imaging 75–90 min after release. Stacks of Spc42-GFP images were acquired every 2 min. Numbers indicate lapsed time. (A) 20-min time-lapse sequence for a WT cell. (B and C) SPB extension and collapse in two HU-treated rad53-21 cells. (D) Graphs depicting changes in spindle length over time for four WT and three rad53-21 cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171834&req=5

fig2: Spindle dynamics in rad53-21 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP strains were released from G1 into 200 mM HU. Cells were prepared for imaging 75–90 min after release. Stacks of Spc42-GFP images were acquired every 2 min. Numbers indicate lapsed time. (A) 20-min time-lapse sequence for a WT cell. (B and C) SPB extension and collapse in two HU-treated rad53-21 cells. (D) Graphs depicting changes in spindle length over time for four WT and three rad53-21 cells.
Mentions: We continued our analysis of the rad53 S phase checkpoint defect by visualizing spindle extension using live cell imaging (Videos 1–5, available at http://www.jcb.org/cgi/content/full/jcb.200412076/DC1). WT and rad53-21 SPC42-GFP strains were released from G1 arrest into 200 mM HU media. Filming was initiated once spindle extension was occurring in ∼25% of the population, allowing visualization of the initial extension of the spindle in some cells and subsequent changes once spindle extension had occurred in others. In HU-arrested WT cells, spindle length remained relatively constant at ∼2 μm, although translocations of the spindle within the cell were observed (Fig. 2 A). Spindle translocations were also observed in rad53 mutants, but were accompanied by SPB separation at rates comparable to the 0.5 μm/min observed during the rapid phase of anaphase B (Straight et al., 1997; Fig. 2, B–D). Unexpectedly, spindle extension was in fact reversible, with SPBs contracting at rates similar to the rate of extension. This cycle was variable, with seemingly stochastic conversions between extension and collapse. These dynamics account for the continuum of spindle lengths in HU-treated rad53 cells and are distinct from unidirectional spindle elongation during anaphase.

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