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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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Spindle extension in HU-treated rad53-21 mutants. (A) WT (JBY430) and rad53-21 (JBY1201) GFP-TUB1 TRP1-GFP cells were released from G1 into 200 mM HU and visualized by GFP fluorescence and low intensity bright-field illumination after 90 min. Arrows,TRP1-GFP foci; pointers, reduced tubulin-GFP in rad53 spindles. Bar, 5 μm. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (SPC42-GFP; green) in HU-treated rad53-21 mutants (JBY1274) 2 h after release from G1 into 200 mM HU. Bar, 5 μm. (C) Spindle length in HU-arrested WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP cells. The distance between Spc42-GFP foci was measured in 500 cells 2 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (D) Spindle extension kinetics in WT (Y300) and rad53-21 (Y301) strains. Time points from cultures released from G1 with (right) or without (left) 200 mM HU were processed for FACS and α-tubulin immunofluorescence. The percentage of spindles ≥3 μm (open squares, WT; open circles, rad53-21) and budded cells (hatched square, WT; hatched circle, rad53-21) was determined. (E) Clb2-Cdk1 activity in WT (JBY012; left) and rad53-21 (JBY013; right) cells. Cells harboring Clb2-HA3× were released from G1 in the presence (circles) or absence (squares) of 200 mM HU, and histone H1 kinase activity in α-HA immunoprecipitates was quantified.
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fig1: Spindle extension in HU-treated rad53-21 mutants. (A) WT (JBY430) and rad53-21 (JBY1201) GFP-TUB1 TRP1-GFP cells were released from G1 into 200 mM HU and visualized by GFP fluorescence and low intensity bright-field illumination after 90 min. Arrows,TRP1-GFP foci; pointers, reduced tubulin-GFP in rad53 spindles. Bar, 5 μm. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (SPC42-GFP; green) in HU-treated rad53-21 mutants (JBY1274) 2 h after release from G1 into 200 mM HU. Bar, 5 μm. (C) Spindle length in HU-arrested WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP cells. The distance between Spc42-GFP foci was measured in 500 cells 2 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (D) Spindle extension kinetics in WT (Y300) and rad53-21 (Y301) strains. Time points from cultures released from G1 with (right) or without (left) 200 mM HU were processed for FACS and α-tubulin immunofluorescence. The percentage of spindles ≥3 μm (open squares, WT; open circles, rad53-21) and budded cells (hatched square, WT; hatched circle, rad53-21) was determined. (E) Clb2-Cdk1 activity in WT (JBY012; left) and rad53-21 (JBY013; right) cells. Cells harboring Clb2-HA3× were released from G1 in the presence (circles) or absence (squares) of 200 mM HU, and histone H1 kinase activity in α-HA immunoprecipitates was quantified.

Mentions: We consistently noticed differences between spindle extension in mec1 and rad53 mutants treated with HU and anaphase spindle elongation, prompting a detailed examination of the S phase checkpoint cell cycle arrest defect. The rad53-21 allele was chosen for this analysis because rad53-21 is proficient for the essential function of RAD53 but exhibits an S phase checkpoint defect equivalent to a rad53 deletion (Desany et al., 1998). Wild-type (WT) and rad53-21 cells were synchronized in G1 and released in the presence or absence of 200 mM HU. Spindle morphology was examined using tubulin-GFP and a CEN-proximal GFP chromosome tag (TRP1-GFP; Straight et al., 1997). WT cells arrested with a 1–2-μm spindle and a single TRP1-GFP dot adjacent to one SPB (Fig. 1 A). In contrast, many rad53-21 cells displayed spindle extension, frequently characterized by reduced tubulin intensity in the central spindle, suggesting collapse, breakage, or disassembly. These aberrant spindles were only observed after HU treatment; spindle elongation in rad53-21 cells in the absence of HU was indistinguishable from WT controls (Fig. 1 D and not depicted). During rad53-21 spindle extension, TRP1-GFP was visualized as a single dot closely associated with either SPB (Fig. 1 A); DAPI staining confirmed the majority of DNA was segregated on the spindle (Fig. 1 B). To provide a more quantitative assessment of spindle extension, a GFP-tagged SPB protein was used to measure pole separation in HU-treated rad53-21 mutants. Using 3 μm as a minimum length for an extended spindle, ∼40–50% of rad53-21 cells exhibited spindle extension with similar kinetics to anaphase spindle elongation in an unperturbed cell cycle (Fig. 1, C and D). Most spindles did not extend beyond 5 μm, far shorter than the ∼10-μm spindles characteristic of complete anaphase extension (Fig. 1 C). Thus, spindle extension in HU-treated rad53 mutants is variable, incomplete, and accompanied by perturbations to spindle morphology.


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)

Spindle extension in HU-treated rad53-21 mutants. (A) WT (JBY430) and rad53-21 (JBY1201) GFP-TUB1 TRP1-GFP cells were released from G1 into 200 mM HU and visualized by GFP fluorescence and low intensity bright-field illumination after 90 min. Arrows,TRP1-GFP foci; pointers, reduced tubulin-GFP in rad53 spindles. Bar, 5 μm. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (SPC42-GFP; green) in HU-treated rad53-21 mutants (JBY1274) 2 h after release from G1 into 200 mM HU. Bar, 5 μm. (C) Spindle length in HU-arrested WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP cells. The distance between Spc42-GFP foci was measured in 500 cells 2 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (D) Spindle extension kinetics in WT (Y300) and rad53-21 (Y301) strains. Time points from cultures released from G1 with (right) or without (left) 200 mM HU were processed for FACS and α-tubulin immunofluorescence. The percentage of spindles ≥3 μm (open squares, WT; open circles, rad53-21) and budded cells (hatched square, WT; hatched circle, rad53-21) was determined. (E) Clb2-Cdk1 activity in WT (JBY012; left) and rad53-21 (JBY013; right) cells. Cells harboring Clb2-HA3× were released from G1 in the presence (circles) or absence (squares) of 200 mM HU, and histone H1 kinase activity in α-HA immunoprecipitates was quantified.
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Related In: Results  -  Collection

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fig1: Spindle extension in HU-treated rad53-21 mutants. (A) WT (JBY430) and rad53-21 (JBY1201) GFP-TUB1 TRP1-GFP cells were released from G1 into 200 mM HU and visualized by GFP fluorescence and low intensity bright-field illumination after 90 min. Arrows,TRP1-GFP foci; pointers, reduced tubulin-GFP in rad53 spindles. Bar, 5 μm. (B) Pseudocolored images of chromatin (DAPI; red) and spindle poles (SPC42-GFP; green) in HU-treated rad53-21 mutants (JBY1274) 2 h after release from G1 into 200 mM HU. Bar, 5 μm. (C) Spindle length in HU-arrested WT (JBY1129) and rad53-21 (JBY1274) SPC42-GFP cells. The distance between Spc42-GFP foci was measured in 500 cells 2 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (D) Spindle extension kinetics in WT (Y300) and rad53-21 (Y301) strains. Time points from cultures released from G1 with (right) or without (left) 200 mM HU were processed for FACS and α-tubulin immunofluorescence. The percentage of spindles ≥3 μm (open squares, WT; open circles, rad53-21) and budded cells (hatched square, WT; hatched circle, rad53-21) was determined. (E) Clb2-Cdk1 activity in WT (JBY012; left) and rad53-21 (JBY013; right) cells. Cells harboring Clb2-HA3× were released from G1 in the presence (circles) or absence (squares) of 200 mM HU, and histone H1 kinase activity in α-HA immunoprecipitates was quantified.
Mentions: We consistently noticed differences between spindle extension in mec1 and rad53 mutants treated with HU and anaphase spindle elongation, prompting a detailed examination of the S phase checkpoint cell cycle arrest defect. The rad53-21 allele was chosen for this analysis because rad53-21 is proficient for the essential function of RAD53 but exhibits an S phase checkpoint defect equivalent to a rad53 deletion (Desany et al., 1998). Wild-type (WT) and rad53-21 cells were synchronized in G1 and released in the presence or absence of 200 mM HU. Spindle morphology was examined using tubulin-GFP and a CEN-proximal GFP chromosome tag (TRP1-GFP; Straight et al., 1997). WT cells arrested with a 1–2-μm spindle and a single TRP1-GFP dot adjacent to one SPB (Fig. 1 A). In contrast, many rad53-21 cells displayed spindle extension, frequently characterized by reduced tubulin intensity in the central spindle, suggesting collapse, breakage, or disassembly. These aberrant spindles were only observed after HU treatment; spindle elongation in rad53-21 cells in the absence of HU was indistinguishable from WT controls (Fig. 1 D and not depicted). During rad53-21 spindle extension, TRP1-GFP was visualized as a single dot closely associated with either SPB (Fig. 1 A); DAPI staining confirmed the majority of DNA was segregated on the spindle (Fig. 1 B). To provide a more quantitative assessment of spindle extension, a GFP-tagged SPB protein was used to measure pole separation in HU-treated rad53-21 mutants. Using 3 μm as a minimum length for an extended spindle, ∼40–50% of rad53-21 cells exhibited spindle extension with similar kinetics to anaphase spindle elongation in an unperturbed cell cycle (Fig. 1, C and D). Most spindles did not extend beyond 5 μm, far shorter than the ∼10-μm spindles characteristic of complete anaphase extension (Fig. 1 C). Thus, spindle extension in HU-treated rad53 mutants is variable, incomplete, and accompanied by perturbations to spindle morphology.

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