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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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Dicentric bridging restricts spindle extension in HU-treated rad53 mutants. LEU2-GFP (JBY541), his4::GAL-CEN3 LEU2-GFP (JBY1208), rad53-21 LEU2-GFP (JBY1201), and two rad53-21 his4::GAL-CEN3 LEU2-GFP segregants (JBY1203 and JBY1206) were propagated in galactose media, placed at G1 arrest, and released into 200 mM HU glucose media to activate the dicentric. (A) After 2 h, cells were processed for α-tubulin immunofluorescence and spindle length was measured for 250 cells. The percentage of spindles ≥3 μm is indicated. (B) Starting at 90 min after release, GFP-tagged chromatin between the dicentric CENs was visualized by fluorescence. Bars, 5 μm.
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fig9: Dicentric bridging restricts spindle extension in HU-treated rad53 mutants. LEU2-GFP (JBY541), his4::GAL-CEN3 LEU2-GFP (JBY1208), rad53-21 LEU2-GFP (JBY1201), and two rad53-21 his4::GAL-CEN3 LEU2-GFP segregants (JBY1203 and JBY1206) were propagated in galactose media, placed at G1 arrest, and released into 200 mM HU glucose media to activate the dicentric. (A) After 2 h, cells were processed for α-tubulin immunofluorescence and spindle length was measured for 250 cells. The percentage of spindles ≥3 μm is indicated. (B) Starting at 90 min after release, GFP-tagged chromatin between the dicentric CENs was visualized by fluorescence. Bars, 5 μm.

Mentions: One hypothesis to accommodate our observations is that Rad53 promotes a cohesin-independent form of chromosome bi-orientation by ensuring that CENs replicate during HU challenge. If this hypothesis is correct, bridging a dicentric chromosome toward opposite spindle poles might partially substitute for Rad53 in restraining spindle extension. Conditional dicentrics have been constructed in which KT assembly at an exogenous CEN can be activated by transferring cells from galactose to glucose media (Thrower and Bloom, 2001). By integrating a GFP chromosome tag between the two CENs (LEU2-GFP) on the dicentric it is possible to visualize the bridged chromatin region. We observed that dicentric activation in HU-treated rad53-21 strains did in fact decrease spindle extension beyond 3 μm (Fig. 9 A). This restriction was accompanied by LEU2-GFP becoming extended into a series of punctate foci, suggesting the two KTs were oriented toward opposite poles and placed under tension (Fig. 9 B).


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)

Dicentric bridging restricts spindle extension in HU-treated rad53 mutants. LEU2-GFP (JBY541), his4::GAL-CEN3 LEU2-GFP (JBY1208), rad53-21 LEU2-GFP (JBY1201), and two rad53-21 his4::GAL-CEN3 LEU2-GFP segregants (JBY1203 and JBY1206) were propagated in galactose media, placed at G1 arrest, and released into 200 mM HU glucose media to activate the dicentric. (A) After 2 h, cells were processed for α-tubulin immunofluorescence and spindle length was measured for 250 cells. The percentage of spindles ≥3 μm is indicated. (B) Starting at 90 min after release, GFP-tagged chromatin between the dicentric CENs was visualized by fluorescence. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171834&req=5

fig9: Dicentric bridging restricts spindle extension in HU-treated rad53 mutants. LEU2-GFP (JBY541), his4::GAL-CEN3 LEU2-GFP (JBY1208), rad53-21 LEU2-GFP (JBY1201), and two rad53-21 his4::GAL-CEN3 LEU2-GFP segregants (JBY1203 and JBY1206) were propagated in galactose media, placed at G1 arrest, and released into 200 mM HU glucose media to activate the dicentric. (A) After 2 h, cells were processed for α-tubulin immunofluorescence and spindle length was measured for 250 cells. The percentage of spindles ≥3 μm is indicated. (B) Starting at 90 min after release, GFP-tagged chromatin between the dicentric CENs was visualized by fluorescence. Bars, 5 μm.
Mentions: One hypothesis to accommodate our observations is that Rad53 promotes a cohesin-independent form of chromosome bi-orientation by ensuring that CENs replicate during HU challenge. If this hypothesis is correct, bridging a dicentric chromosome toward opposite spindle poles might partially substitute for Rad53 in restraining spindle extension. Conditional dicentrics have been constructed in which KT assembly at an exogenous CEN can be activated by transferring cells from galactose to glucose media (Thrower and Bloom, 2001). By integrating a GFP chromosome tag between the two CENs (LEU2-GFP) on the dicentric it is possible to visualize the bridged chromatin region. We observed that dicentric activation in HU-treated rad53-21 strains did in fact decrease spindle extension beyond 3 μm (Fig. 9 A). This restriction was accompanied by LEU2-GFP becoming extended into a series of punctate foci, suggesting the two KTs were oriented toward opposite poles and placed under tension (Fig. 9 B).

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