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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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SCC1 is not required to prevent spindle extension during HU arrest. WT (Y300), rad53-21 (Y301), and scc1-73 (JBY585) strains were released from G1 into 200 mM HU at 35°C. cdc23-1 (JBY622), cdc23-1rad53-21 (JBY623), and cdc23-1scc1-73 (JBY1305) strains were released at 35°C in the absence of HU. (A) Spindle (α-tubulin) and chromosome (DAPI) morphology in HU- or cdc23-arrested strains 2.5 h after G1 release. Bars, 5 μm. (B) Kinetics of spindle extension and budding. (left) HU-arrested strains (squares, WT; circles, rad53-21; triangles, scc1-73). (right) cdc23-1–arrested strains (squares, cdc23-1; circles, cdc23-1rad53-21; triangles, cdc23-1scc1-73).
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fig3: SCC1 is not required to prevent spindle extension during HU arrest. WT (Y300), rad53-21 (Y301), and scc1-73 (JBY585) strains were released from G1 into 200 mM HU at 35°C. cdc23-1 (JBY622), cdc23-1rad53-21 (JBY623), and cdc23-1scc1-73 (JBY1305) strains were released at 35°C in the absence of HU. (A) Spindle (α-tubulin) and chromosome (DAPI) morphology in HU- or cdc23-arrested strains 2.5 h after G1 release. Bars, 5 μm. (B) Kinetics of spindle extension and budding. (left) HU-arrested strains (squares, WT; circles, rad53-21; triangles, scc1-73). (right) cdc23-1–arrested strains (squares, cdc23-1; circles, cdc23-1rad53-21; triangles, cdc23-1scc1-73).

Mentions: Yeast mutants defective for APC-mediated Pds1 degradation cannot disjoin chromatids and arrest with an ∼2–3-μm metaphase spindle. The importance of cohesion in preventing spindle extension can be observed using APC mutants that are also defective for cohesion, as these cells undergo spindle extension even though Pds1 is stabilized (Michaelis et al., 1997). Although Pds1 is not required to prevent spindle extension during S phase checkpoint arrest, cohesin is deposited on chromosomes at an HU replication block (Blat and Kleckner, 1999). Thus, cohesion at early replicating CEN regions could prevent spindle extension at a point when Mcd1/Scc1 was not yet susceptible to cleavage. Alternatively, S phase checkpoint arrest could occur through a cohesin-independent mechanism. To distinguish between these scenarios, we compared the ability of cohesion-defective scc1-73 mutants to restrain spindle extension during either S phase checkpoint arrest induced by HU or during metaphase arrest induced by inactivation of the APC component Cdc23. WT, rad53-21, and scc1-73 mutants were released from a G1 block into 200 mM HU media at a scc1-73 nonpermissive temperature. Whereas rad53-21 initiated spindle extension between 60–90 min, scc1-73 mutants arrested with normal preanaphase spindles (Fig. 3; Guacci et al., 1997). However, when cdc23-1, cdc23-1rad53-21, and cdc23-1scc1-73 cells were released in the absence of HU, we observed a reciprocal pattern where cdc23rad53-21 mutants arrested normally but cdc23-1scc1-73 exhibited spindle extension. Thus, Mcd1/Scc1 does not couple spindle extension to anaphase onset during S phase checkpoint arrest.


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)

SCC1 is not required to prevent spindle extension during HU arrest. WT (Y300), rad53-21 (Y301), and scc1-73 (JBY585) strains were released from G1 into 200 mM HU at 35°C. cdc23-1 (JBY622), cdc23-1rad53-21 (JBY623), and cdc23-1scc1-73 (JBY1305) strains were released at 35°C in the absence of HU. (A) Spindle (α-tubulin) and chromosome (DAPI) morphology in HU- or cdc23-arrested strains 2.5 h after G1 release. Bars, 5 μm. (B) Kinetics of spindle extension and budding. (left) HU-arrested strains (squares, WT; circles, rad53-21; triangles, scc1-73). (right) cdc23-1–arrested strains (squares, cdc23-1; circles, cdc23-1rad53-21; triangles, cdc23-1scc1-73).
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fig3: SCC1 is not required to prevent spindle extension during HU arrest. WT (Y300), rad53-21 (Y301), and scc1-73 (JBY585) strains were released from G1 into 200 mM HU at 35°C. cdc23-1 (JBY622), cdc23-1rad53-21 (JBY623), and cdc23-1scc1-73 (JBY1305) strains were released at 35°C in the absence of HU. (A) Spindle (α-tubulin) and chromosome (DAPI) morphology in HU- or cdc23-arrested strains 2.5 h after G1 release. Bars, 5 μm. (B) Kinetics of spindle extension and budding. (left) HU-arrested strains (squares, WT; circles, rad53-21; triangles, scc1-73). (right) cdc23-1–arrested strains (squares, cdc23-1; circles, cdc23-1rad53-21; triangles, cdc23-1scc1-73).
Mentions: Yeast mutants defective for APC-mediated Pds1 degradation cannot disjoin chromatids and arrest with an ∼2–3-μm metaphase spindle. The importance of cohesion in preventing spindle extension can be observed using APC mutants that are also defective for cohesion, as these cells undergo spindle extension even though Pds1 is stabilized (Michaelis et al., 1997). Although Pds1 is not required to prevent spindle extension during S phase checkpoint arrest, cohesin is deposited on chromosomes at an HU replication block (Blat and Kleckner, 1999). Thus, cohesion at early replicating CEN regions could prevent spindle extension at a point when Mcd1/Scc1 was not yet susceptible to cleavage. Alternatively, S phase checkpoint arrest could occur through a cohesin-independent mechanism. To distinguish between these scenarios, we compared the ability of cohesion-defective scc1-73 mutants to restrain spindle extension during either S phase checkpoint arrest induced by HU or during metaphase arrest induced by inactivation of the APC component Cdc23. WT, rad53-21, and scc1-73 mutants were released from a G1 block into 200 mM HU media at a scc1-73 nonpermissive temperature. Whereas rad53-21 initiated spindle extension between 60–90 min, scc1-73 mutants arrested with normal preanaphase spindles (Fig. 3; Guacci et al., 1997). However, when cdc23-1, cdc23-1rad53-21, and cdc23-1scc1-73 cells were released in the absence of HU, we observed a reciprocal pattern where cdc23rad53-21 mutants arrested normally but cdc23-1scc1-73 exhibited spindle extension. Thus, Mcd1/Scc1 does not couple spindle extension to anaphase onset during S phase checkpoint arrest.

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