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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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Analysis of Esh− mutants. (A) Cell survival after HU treatment. Cultures of WT (Y300; squares), smt4-3 (JBY047; triangles), ask1-1 (JBY368; circles), and rad53-21 (Y301; diamonds) cells were exposed to 200 mM HU. At the indicated times, aliquots were plated for cell viability. (B) Spindle length during HU arrest. The distance between Spc42-GFP foci was measured for 250 WT (JBY1129), smt4-3 (JBY1312), and ask1-1 (JBY1196) SPC42-GFP cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) MIF2 clones isolated as Smt4 two-hybrid interactors. Regions of similarity to CENP-C (black boxes) and the HMGI (Y) domain (gray box) are indicated. pDAB (GAL4 DNA binding domain [DBD] vector), pACT (GAL4 activation domain [AD] vector), pSE1111 (SNF4-DBD), pJBN84 (SMT4-DBD), and MIF2-AD clones p20.1, p53.1, and p59.1 were analyzed in the indicated combinations. Growth on Trp−Leu−Ade− media indicates a two-hybrid interaction.
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fig4: Analysis of Esh− mutants. (A) Cell survival after HU treatment. Cultures of WT (Y300; squares), smt4-3 (JBY047; triangles), ask1-1 (JBY368; circles), and rad53-21 (Y301; diamonds) cells were exposed to 200 mM HU. At the indicated times, aliquots were plated for cell viability. (B) Spindle length during HU arrest. The distance between Spc42-GFP foci was measured for 250 WT (JBY1129), smt4-3 (JBY1312), and ask1-1 (JBY1196) SPC42-GFP cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) MIF2 clones isolated as Smt4 two-hybrid interactors. Regions of similarity to CENP-C (black boxes) and the HMGI (Y) domain (gray box) are indicated. pDAB (GAL4 DNA binding domain [DBD] vector), pACT (GAL4 activation domain [AD] vector), pSE1111 (SNF4-DBD), pJBN84 (SMT4-DBD), and MIF2-AD clones p20.1, p53.1, and p59.1 were analyzed in the indicated combinations. Growth on Trp−Leu−Ade− media indicates a two-hybrid interaction.

Mentions: To define the cohesin-independent forces restraining spindle extension during S phase, we conducted a genetic screen for mutants exhibiting HU sensitivity and extended spindles after HU treatment (Esh− phenotype; Alcasabas et al., 2001). Interestingly, mutant alleles of two genes implicated in KT function (ASK1 and SMT4/ULP2) were identified. ASK1 encodes a component of the CEN-binding Dam1/DDD/DASH complex (Li et al., 2002), whereas SMT4 encodes an isopeptidase that deconjugates the ubiquitin-like SUMO protein (Li and Hochstrasser, 2000). SMT4 was isolated as a suppressor of a mutation in the KT protein Mif2, suggesting SMT4 might function in a pathway regulating Mif2 function (Meluh and Koshland, 1995). Indeed, we identified Mif2 as a Smt4 binding partner in a two-hybrid screen, providing another connection between these proteins (Fig. 4 C). smt4-3 and ask1-1 mutants isolated in the Esh− screen failed to recover from HU treatment, although the severity of this defect was less than that of rad53-21 (Fig. 4 A). Using the criteria established for rad53, ∼25% of smt4-3 SPC42-GFP cells and ∼10% of ask1-1 SPC42-GFP cells released from G1 into 200 mM HU media showed an extended spindle phenotype (Fig. 4 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)

Analysis of Esh− mutants. (A) Cell survival after HU treatment. Cultures of WT (Y300; squares), smt4-3 (JBY047; triangles), ask1-1 (JBY368; circles), and rad53-21 (Y301; diamonds) cells were exposed to 200 mM HU. At the indicated times, aliquots were plated for cell viability. (B) Spindle length during HU arrest. The distance between Spc42-GFP foci was measured for 250 WT (JBY1129), smt4-3 (JBY1312), and ask1-1 (JBY1196) SPC42-GFP cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) MIF2 clones isolated as Smt4 two-hybrid interactors. Regions of similarity to CENP-C (black boxes) and the HMGI (Y) domain (gray box) are indicated. pDAB (GAL4 DNA binding domain [DBD] vector), pACT (GAL4 activation domain [AD] vector), pSE1111 (SNF4-DBD), pJBN84 (SMT4-DBD), and MIF2-AD clones p20.1, p53.1, and p59.1 were analyzed in the indicated combinations. Growth on Trp−Leu−Ade− media indicates a two-hybrid interaction.
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Related In: Results  -  Collection

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fig4: Analysis of Esh− mutants. (A) Cell survival after HU treatment. Cultures of WT (Y300; squares), smt4-3 (JBY047; triangles), ask1-1 (JBY368; circles), and rad53-21 (Y301; diamonds) cells were exposed to 200 mM HU. At the indicated times, aliquots were plated for cell viability. (B) Spindle length during HU arrest. The distance between Spc42-GFP foci was measured for 250 WT (JBY1129), smt4-3 (JBY1312), and ask1-1 (JBY1196) SPC42-GFP cells 2.5 h after release from G1 into 200 mM HU. The percentage of spindles ≥3 μm is indicated. (C) MIF2 clones isolated as Smt4 two-hybrid interactors. Regions of similarity to CENP-C (black boxes) and the HMGI (Y) domain (gray box) are indicated. pDAB (GAL4 DNA binding domain [DBD] vector), pACT (GAL4 activation domain [AD] vector), pSE1111 (SNF4-DBD), pJBN84 (SMT4-DBD), and MIF2-AD clones p20.1, p53.1, and p59.1 were analyzed in the indicated combinations. Growth on Trp−Leu−Ade− media indicates a two-hybrid interaction.
Mentions: To define the cohesin-independent forces restraining spindle extension during S phase, we conducted a genetic screen for mutants exhibiting HU sensitivity and extended spindles after HU treatment (Esh− phenotype; Alcasabas et al., 2001). Interestingly, mutant alleles of two genes implicated in KT function (ASK1 and SMT4/ULP2) were identified. ASK1 encodes a component of the CEN-binding Dam1/DDD/DASH complex (Li et al., 2002), whereas SMT4 encodes an isopeptidase that deconjugates the ubiquitin-like SUMO protein (Li and Hochstrasser, 2000). SMT4 was isolated as a suppressor of a mutation in the KT protein Mif2, suggesting SMT4 might function in a pathway regulating Mif2 function (Meluh and Koshland, 1995). Indeed, we identified Mif2 as a Smt4 binding partner in a two-hybrid screen, providing another connection between these proteins (Fig. 4 C). smt4-3 and ask1-1 mutants isolated in the Esh− screen failed to recover from HU treatment, although the severity of this defect was less than that of rad53-21 (Fig. 4 A). Using the criteria established for rad53, ∼25% of smt4-3 SPC42-GFP cells and ∼10% of ask1-1 SPC42-GFP cells released from G1 into 200 mM HU media showed an extended spindle phenotype (Fig. 4 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