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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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MIF2 and ASK1 restrain spindle extension during HU arrest. (A) Cell survival in HU-treated mif2-2 mutants. Cultures of WT (Y300) and mif2-2 (JBY358) strains were treated ± 200 mM HU at 24 or 35°C. Aliquots were plated to monitor cell viability. Squares, WT/24°C/HU; triangles, WT/35°C/HU; diamonds, mif2-2/24°C; circles, mif2-2/24°C/HU; hatched diamonds, mif2-2/35°C/HU; hatched squares, mif2-2/35°C. (B) Spindle length was measured for 250 WT SPC42-GFP (JBY1129), ask1-3 SPC42-GFP (JBY1325), and mif2-2 (JBY358) cells 2.5 h after G1 release into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) Kinetics of spindle extension during HU arrest. WT (Y300, squares), rad53-21 (Y301; diamonds), mif2-2 (JBY358; triangles), and ask1-3 (JBY1325; circles) were released from G1 into 200 mM HU at 35°C. Time points were processed for DAPI and α-tubulin staining.
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fig5: MIF2 and ASK1 restrain spindle extension during HU arrest. (A) Cell survival in HU-treated mif2-2 mutants. Cultures of WT (Y300) and mif2-2 (JBY358) strains were treated ± 200 mM HU at 24 or 35°C. Aliquots were plated to monitor cell viability. Squares, WT/24°C/HU; triangles, WT/35°C/HU; diamonds, mif2-2/24°C; circles, mif2-2/24°C/HU; hatched diamonds, mif2-2/35°C/HU; hatched squares, mif2-2/35°C. (B) Spindle length was measured for 250 WT SPC42-GFP (JBY1129), ask1-3 SPC42-GFP (JBY1325), and mif2-2 (JBY358) cells 2.5 h after G1 release into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) Kinetics of spindle extension during HU arrest. WT (Y300, squares), rad53-21 (Y301; diamonds), mif2-2 (JBY358; triangles), and ask1-3 (JBY1325; circles) were released from G1 into 200 mM HU at 35°C. Time points were processed for DAPI and α-tubulin staining.

Mentions: The results from the Esh− screen are consistent with a role for the KT in preventing spindle extension during HU arrest. However, spindle extension in HU-treated smt4-3 mutants could reflect SUMO regulation of processes unrelated to KT function. Also, the spindle extension phenotype of ask1-1 is less severe than rad53-21. Therefore, we began a more comprehensive analysis of KT-defective strains, beginning with mif2-2 and ask1-3 mutants. At a nonpermissive temperature, ask1-3 cells undergo an aberrant mitosis in which spindles extend in the absence of chromatid disjunction (Li et al., 2002). The failure of chromatids to separate reflects activation of the spindle checkpoint because the delay in sister separation is eliminated in checkpoint-defective ask1-3mad2-Δ mutants. Similarly, when mif2-2 TRP1-GFP cells were released from G1 at a nonpermissive temperature, spindle extension was initiated well in advance of TRP1-GFP disjunction, and the relative timing of chromatid separation and spindle extension was restored in a mif2-2mad2-Δ strain (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200412076/DC1). mif2-2 mutants showed reduced viability when exposed to HU (Fig. 5 A), and, after release from G1 into 200 mM HU media at a nonpermissive temperature, ∼40–50% of mif2-2 and ask1-3 cells displayed a robust Esh− phenotype (Fig. 5 B). Spindle extension was initiated with similar timing to rad53 controls (Fig. 5 C), and, as observed in rad53 mutants, elongated spindles exhibited diminished tubulin staining in interpolar regions (Fig. 6 A). Thus, Ask1 and Mif2 are required to couple spindle extension to anaphase onset not only during a metaphase block induced by the spindle checkpoint but also during an S phase block induced by HU. This finding is in contrast to the requirements for Mcd1/Scc1, which is only manifested once the bulk of S phase is complete, and for Rad53, which is only observed during S phase arrest (Fig. 3).


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)

MIF2 and ASK1 restrain spindle extension during HU arrest. (A) Cell survival in HU-treated mif2-2 mutants. Cultures of WT (Y300) and mif2-2 (JBY358) strains were treated ± 200 mM HU at 24 or 35°C. Aliquots were plated to monitor cell viability. Squares, WT/24°C/HU; triangles, WT/35°C/HU; diamonds, mif2-2/24°C; circles, mif2-2/24°C/HU; hatched diamonds, mif2-2/35°C/HU; hatched squares, mif2-2/35°C. (B) Spindle length was measured for 250 WT SPC42-GFP (JBY1129), ask1-3 SPC42-GFP (JBY1325), and mif2-2 (JBY358) cells 2.5 h after G1 release into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) Kinetics of spindle extension during HU arrest. WT (Y300, squares), rad53-21 (Y301; diamonds), mif2-2 (JBY358; triangles), and ask1-3 (JBY1325; circles) were released from G1 into 200 mM HU at 35°C. Time points were processed for DAPI and α-tubulin staining.
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fig5: MIF2 and ASK1 restrain spindle extension during HU arrest. (A) Cell survival in HU-treated mif2-2 mutants. Cultures of WT (Y300) and mif2-2 (JBY358) strains were treated ± 200 mM HU at 24 or 35°C. Aliquots were plated to monitor cell viability. Squares, WT/24°C/HU; triangles, WT/35°C/HU; diamonds, mif2-2/24°C; circles, mif2-2/24°C/HU; hatched diamonds, mif2-2/35°C/HU; hatched squares, mif2-2/35°C. (B) Spindle length was measured for 250 WT SPC42-GFP (JBY1129), ask1-3 SPC42-GFP (JBY1325), and mif2-2 (JBY358) cells 2.5 h after G1 release into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) Kinetics of spindle extension during HU arrest. WT (Y300, squares), rad53-21 (Y301; diamonds), mif2-2 (JBY358; triangles), and ask1-3 (JBY1325; circles) were released from G1 into 200 mM HU at 35°C. Time points were processed for DAPI and α-tubulin staining.
Mentions: The results from the Esh− screen are consistent with a role for the KT in preventing spindle extension during HU arrest. However, spindle extension in HU-treated smt4-3 mutants could reflect SUMO regulation of processes unrelated to KT function. Also, the spindle extension phenotype of ask1-1 is less severe than rad53-21. Therefore, we began a more comprehensive analysis of KT-defective strains, beginning with mif2-2 and ask1-3 mutants. At a nonpermissive temperature, ask1-3 cells undergo an aberrant mitosis in which spindles extend in the absence of chromatid disjunction (Li et al., 2002). The failure of chromatids to separate reflects activation of the spindle checkpoint because the delay in sister separation is eliminated in checkpoint-defective ask1-3mad2-Δ mutants. Similarly, when mif2-2 TRP1-GFP cells were released from G1 at a nonpermissive temperature, spindle extension was initiated well in advance of TRP1-GFP disjunction, and the relative timing of chromatid separation and spindle extension was restored in a mif2-2mad2-Δ strain (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200412076/DC1). mif2-2 mutants showed reduced viability when exposed to HU (Fig. 5 A), and, after release from G1 into 200 mM HU media at a nonpermissive temperature, ∼40–50% of mif2-2 and ask1-3 cells displayed a robust Esh− phenotype (Fig. 5 B). Spindle extension was initiated with similar timing to rad53 controls (Fig. 5 C), and, as observed in rad53 mutants, elongated spindles exhibited diminished tubulin staining in interpolar regions (Fig. 6 A). Thus, Ask1 and Mif2 are required to couple spindle extension to anaphase onset not only during a metaphase block induced by the spindle checkpoint but also during an S phase block induced by HU. This finding is in contrast to the requirements for Mcd1/Scc1, which is only manifested once the bulk of S phase is complete, and for Rad53, which is only observed during S phase arrest (Fig. 3).

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