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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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pCENARS minichromosomes suppress spindle extension in HU-treated rad53 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP-TRP1 strains were transformed with one (pRS413), two (pRS413 and pRS415), or three (pRS413, pRS415, and pRS416) pCENARS plasmids (Sikorski and Hieter, 1989). WT (Y300) and rad53-21 (Y301) were transformed with four pCENARS plasmids (pRS413, pRS414, pRS415, and pRS416). Transformants were cultured to maintain the plasmids, arrested in G1, and released into yeast extract/peptone/dextrose media containing 200 mM HU. Spindle length was measured in 200 cells at the indicated times using Spc42-GFP foci or α-tubulin immunofluorescence. (A) Effect of pCENARS dosage on spindle extension in HU-treated rad53 mutants (right). (left) Budding kinetics. (B) Spindle length distribution in WT and rad53 mutants after 2.5 h of HU treatment. The percentage of spindles ≥3 μm is indicated. (C) Requirements for pCENARS suppression. WT (JBY1129), rad53-21 (JBY1274), ndc80-1 (JBY1359), and ndc80-1rad53-21 (JBY1400) SPC42-GFP strains and dbf4-1 (JBY997), dbf4-1rad53-21 (JBY1001), mad2-Δ (JBY1393), mad2-Δrad53-21 (JBY1395), scc1-73 (JBY585), and scc1-73rad53-21 (JBY1397) strains ± three pCENARS plasmids were released from G1 into 200 mM HU at 35°C. After 2.5 h, the percentage of spindles ≥3 μm was determined for 200 cells.
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fig10: pCENARS minichromosomes suppress spindle extension in HU-treated rad53 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP-TRP1 strains were transformed with one (pRS413), two (pRS413 and pRS415), or three (pRS413, pRS415, and pRS416) pCENARS plasmids (Sikorski and Hieter, 1989). WT (Y300) and rad53-21 (Y301) were transformed with four pCENARS plasmids (pRS413, pRS414, pRS415, and pRS416). Transformants were cultured to maintain the plasmids, arrested in G1, and released into yeast extract/peptone/dextrose media containing 200 mM HU. Spindle length was measured in 200 cells at the indicated times using Spc42-GFP foci or α-tubulin immunofluorescence. (A) Effect of pCENARS dosage on spindle extension in HU-treated rad53 mutants (right). (left) Budding kinetics. (B) Spindle length distribution in WT and rad53 mutants after 2.5 h of HU treatment. The percentage of spindles ≥3 μm is indicated. (C) Requirements for pCENARS suppression. WT (JBY1129), rad53-21 (JBY1274), ndc80-1 (JBY1359), and ndc80-1rad53-21 (JBY1400) SPC42-GFP strains and dbf4-1 (JBY997), dbf4-1rad53-21 (JBY1001), mad2-Δ (JBY1393), mad2-Δrad53-21 (JBY1395), scc1-73 (JBY585), and scc1-73rad53-21 (JBY1397) strains ± three pCENARS plasmids were released from G1 into 200 mM HU at 35°C. After 2.5 h, the percentage of spindles ≥3 μm was determined for 200 cells.

Mentions: Although budding yeast CENs replicate early in S phase, the origins from which these forks originate are typically located several kilobases away from the CEN (Yabuki et al., 2002). If Rad53 promotes CEN replication during HU treatment by allowing forks to traverse the distance to the CEN without collapsing, we reasoned that reducing this distance might compensate for loss of Rad53 function. Fortuitously, such a juxtaposition of CEN and origin sequences is found on circular minichromosome plasmids engineered for transfer of DNA sequences in yeast (Sikorski and Hieter, 1989). The origin sequence (ARS) on these minichromosomes is only ∼300 bp distant from the CEN. Therefore, we examined the consequences of introducing one, two, three, and four CENARS minichromosomes into HU-treated rad53-21 cells. Remarkably, as few as two CENARS minichromosomes reduced spindle extension in HU-treated rad53-21 mutants by ∼50%, and a further decrease was seen in cells harboring three or four minichromosomes (Fig. 10, A and B). If these minichromosomes suppress rad53 spindle extension by allowing CEN replication, the suppression should require DNA replication and KT assembly. Indeed, the suppression afforded by three CENARS minichromosomes after HU treatment was not observed in dbf4-1 and dbf4-1rad53-21 cells defective for initiation of DNA replication or in ndc80-1 and ndc80-1rad53-21 cells (Fig. 10 C). In contrast, CENARS minichromosomes were still able to suppress spindle extension in HU-treated scc1-73rad53-21 and mad2-Δrad53-21 strains, indicating this effect did not require cohesion or spindle checkpoint activation. Therefore, reducing the distance between origins of replication and a critical number of CENs is sufficient to largely restrain spindle extension in the absence of Rad53 signaling.


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)

pCENARS minichromosomes suppress spindle extension in HU-treated rad53 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP-TRP1 strains were transformed with one (pRS413), two (pRS413 and pRS415), or three (pRS413, pRS415, and pRS416) pCENARS plasmids (Sikorski and Hieter, 1989). WT (Y300) and rad53-21 (Y301) were transformed with four pCENARS plasmids (pRS413, pRS414, pRS415, and pRS416). Transformants were cultured to maintain the plasmids, arrested in G1, and released into yeast extract/peptone/dextrose media containing 200 mM HU. Spindle length was measured in 200 cells at the indicated times using Spc42-GFP foci or α-tubulin immunofluorescence. (A) Effect of pCENARS dosage on spindle extension in HU-treated rad53 mutants (right). (left) Budding kinetics. (B) Spindle length distribution in WT and rad53 mutants after 2.5 h of HU treatment. The percentage of spindles ≥3 μm is indicated. (C) Requirements for pCENARS suppression. WT (JBY1129), rad53-21 (JBY1274), ndc80-1 (JBY1359), and ndc80-1rad53-21 (JBY1400) SPC42-GFP strains and dbf4-1 (JBY997), dbf4-1rad53-21 (JBY1001), mad2-Δ (JBY1393), mad2-Δrad53-21 (JBY1395), scc1-73 (JBY585), and scc1-73rad53-21 (JBY1397) strains ± three pCENARS plasmids were released from G1 into 200 mM HU at 35°C. After 2.5 h, the percentage of spindles ≥3 μm was determined for 200 cells.
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fig10: pCENARS minichromosomes suppress spindle extension in HU-treated rad53 mutants. WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP-TRP1 strains were transformed with one (pRS413), two (pRS413 and pRS415), or three (pRS413, pRS415, and pRS416) pCENARS plasmids (Sikorski and Hieter, 1989). WT (Y300) and rad53-21 (Y301) were transformed with four pCENARS plasmids (pRS413, pRS414, pRS415, and pRS416). Transformants were cultured to maintain the plasmids, arrested in G1, and released into yeast extract/peptone/dextrose media containing 200 mM HU. Spindle length was measured in 200 cells at the indicated times using Spc42-GFP foci or α-tubulin immunofluorescence. (A) Effect of pCENARS dosage on spindle extension in HU-treated rad53 mutants (right). (left) Budding kinetics. (B) Spindle length distribution in WT and rad53 mutants after 2.5 h of HU treatment. The percentage of spindles ≥3 μm is indicated. (C) Requirements for pCENARS suppression. WT (JBY1129), rad53-21 (JBY1274), ndc80-1 (JBY1359), and ndc80-1rad53-21 (JBY1400) SPC42-GFP strains and dbf4-1 (JBY997), dbf4-1rad53-21 (JBY1001), mad2-Δ (JBY1393), mad2-Δrad53-21 (JBY1395), scc1-73 (JBY585), and scc1-73rad53-21 (JBY1397) strains ± three pCENARS plasmids were released from G1 into 200 mM HU at 35°C. After 2.5 h, the percentage of spindles ≥3 μm was determined for 200 cells.
Mentions: Although budding yeast CENs replicate early in S phase, the origins from which these forks originate are typically located several kilobases away from the CEN (Yabuki et al., 2002). If Rad53 promotes CEN replication during HU treatment by allowing forks to traverse the distance to the CEN without collapsing, we reasoned that reducing this distance might compensate for loss of Rad53 function. Fortuitously, such a juxtaposition of CEN and origin sequences is found on circular minichromosome plasmids engineered for transfer of DNA sequences in yeast (Sikorski and Hieter, 1989). The origin sequence (ARS) on these minichromosomes is only ∼300 bp distant from the CEN. Therefore, we examined the consequences of introducing one, two, three, and four CENARS minichromosomes into HU-treated rad53-21 cells. Remarkably, as few as two CENARS minichromosomes reduced spindle extension in HU-treated rad53-21 mutants by ∼50%, and a further decrease was seen in cells harboring three or four minichromosomes (Fig. 10, A and B). If these minichromosomes suppress rad53 spindle extension by allowing CEN replication, the suppression should require DNA replication and KT assembly. Indeed, the suppression afforded by three CENARS minichromosomes after HU treatment was not observed in dbf4-1 and dbf4-1rad53-21 cells defective for initiation of DNA replication or in ndc80-1 and ndc80-1rad53-21 cells (Fig. 10 C). In contrast, CENARS minichromosomes were still able to suppress spindle extension in HU-treated scc1-73rad53-21 and mad2-Δrad53-21 strains, indicating this effect did not require cohesion or spindle checkpoint activation. Therefore, reducing the distance between origins of replication and a critical number of CENs is sufficient to largely restrain spindle extension in the absence of Rad53 signaling.

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