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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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HU-treated mad2 and ipl1 mutants. (A) Spindle length was measured in 250 WT (Y300), rad53-21 (Y301), mad2-Δ (JBY1393), and rad53-21mad2-Δ (JBY1395) cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (Spc42-GFP; green) in HU-treated WT (JBY1129), rad53-21 (JBY1274), ipl1-321 (JBY1353), and ipl1-321rad53-21 (JBY1389) SPC42-GFP strains 2.5 h after G1 release into 200 mM HU. Bar, 5 μm.
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fig8: HU-treated mad2 and ipl1 mutants. (A) Spindle length was measured in 250 WT (Y300), rad53-21 (Y301), mad2-Δ (JBY1393), and rad53-21mad2-Δ (JBY1395) cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (Spc42-GFP; green) in HU-treated WT (JBY1129), rad53-21 (JBY1274), ipl1-321 (JBY1353), and ipl1-321rad53-21 (JBY1389) SPC42-GFP strains 2.5 h after G1 release into 200 mM HU. Bar, 5 μm.

Mentions: The aforementioned approach was extended to include mutants defective for the Aurora B homologue Ipl1. Because rad53 mutants did not appear defective for KT–spindle attachment, the role of Ipl1 was of particular interest because Ipl1 mediates two functions that, although not directly required for chromosomes to connect to the spindle, play important roles in facilitating KT bi-orientation. First, Ipl1 controls a spindle checkpoint response that delays Pds1 proteolysis when KTs are not tensed during spindle attachment (Biggins and Murray, 2001). It is unlikely that this aspect of Ipl1 function prevents spindle extension during HU arrest because checkpoint-defective mad2-Δ mutants arrested with normal preanaphase spindles after HU treatment (Fig. 8 A). Ipl1 also promotes bi-orientation by destabilizing monopolar chromatid connections. This function has been illustrated in cdc6 mutants, which fail to initiate DNA replication and undergo a “reductional” anaphase where unreplicated chromosomes segregate with both poles during spindle extension (Piatti et al., 1995). Chromosomes only associate with a single pole in cdc6ipl1 double mutants, revealing Ipl1 releases KTs from their initial SPB attachment and redistributes them to both poles during spindle assembly (Tanaka et al., 2002). Consistent with a role for bi-orientation in generating the traction required to restrain spindle extension, cdc23ip1-321 mutants displayed extended spindles both during metaphase arrest and after HU treatment (Fig. 7). Furthermore, chromatin remained predominately associated with a single pole during spindle extension in HU-treated ipl1-321 and rad53ipl1-321 SPC42-GFP cells (Fig. 8 B). (To observe extension in ipl1-321 strains it was necessary to preshift the cells to 35°C for 1 h before G1 release.) First, we conclude that Ipl1 is required for chromosome segregation in HU-treated rad53 cells; and, second, that a distribution of KT–MT attachments to both spindle poles is necessary to prevent spindle extension 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)

HU-treated mad2 and ipl1 mutants. (A) Spindle length was measured in 250 WT (Y300), rad53-21 (Y301), mad2-Δ (JBY1393), and rad53-21mad2-Δ (JBY1395) cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (Spc42-GFP; green) in HU-treated WT (JBY1129), rad53-21 (JBY1274), ipl1-321 (JBY1353), and ipl1-321rad53-21 (JBY1389) SPC42-GFP strains 2.5 h after G1 release into 200 mM HU. Bar, 5 μm.
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fig8: HU-treated mad2 and ipl1 mutants. (A) Spindle length was measured in 250 WT (Y300), rad53-21 (Y301), mad2-Δ (JBY1393), and rad53-21mad2-Δ (JBY1395) cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (Spc42-GFP; green) in HU-treated WT (JBY1129), rad53-21 (JBY1274), ipl1-321 (JBY1353), and ipl1-321rad53-21 (JBY1389) SPC42-GFP strains 2.5 h after G1 release into 200 mM HU. Bar, 5 μm.
Mentions: The aforementioned approach was extended to include mutants defective for the Aurora B homologue Ipl1. Because rad53 mutants did not appear defective for KT–spindle attachment, the role of Ipl1 was of particular interest because Ipl1 mediates two functions that, although not directly required for chromosomes to connect to the spindle, play important roles in facilitating KT bi-orientation. First, Ipl1 controls a spindle checkpoint response that delays Pds1 proteolysis when KTs are not tensed during spindle attachment (Biggins and Murray, 2001). It is unlikely that this aspect of Ipl1 function prevents spindle extension during HU arrest because checkpoint-defective mad2-Δ mutants arrested with normal preanaphase spindles after HU treatment (Fig. 8 A). Ipl1 also promotes bi-orientation by destabilizing monopolar chromatid connections. This function has been illustrated in cdc6 mutants, which fail to initiate DNA replication and undergo a “reductional” anaphase where unreplicated chromosomes segregate with both poles during spindle extension (Piatti et al., 1995). Chromosomes only associate with a single pole in cdc6ipl1 double mutants, revealing Ipl1 releases KTs from their initial SPB attachment and redistributes them to both poles during spindle assembly (Tanaka et al., 2002). Consistent with a role for bi-orientation in generating the traction required to restrain spindle extension, cdc23ip1-321 mutants displayed extended spindles both during metaphase arrest and after HU treatment (Fig. 7). Furthermore, chromatin remained predominately associated with a single pole during spindle extension in HU-treated ipl1-321 and rad53ipl1-321 SPC42-GFP cells (Fig. 8 B). (To observe extension in ipl1-321 strains it was necessary to preshift the cells to 35°C for 1 h before G1 release.) First, we conclude that Ipl1 is required for chromosome segregation in HU-treated rad53 cells; and, second, that a distribution of KT–MT attachments to both spindle poles is necessary to prevent spindle extension 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