scholarly journals MRX (Mre11/Rad50/Xrs2) Mutants Reveal Dual Intra-S-Phase Checkpoint Systems in Budding Yeast

Cell Cycle ◽  
2005 ◽  
Vol 4 (8) ◽  
pp. 4073-4077 ◽  
Author(s):  
Catherine A. Andrews ◽  
Duncan J. Clarke
Keyword(s):  
Budding Yeast ◽  
S Phase ◽  
S Phase Checkpoint

scholarly journals Inhibition of spindle extension through the yeast S phase checkpoint is coupled to replication fork stability and the integrity of centromeric DNA

2019 ◽  
Vol 30 (22) ◽  
pp. 2771-2789
Author(s):  
Jeff Julius ◽  
Jie Peng ◽  
Andrew McCulley ◽  
Chris Caridi ◽  
Remigiusz Arnak ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Replication Fork ◽  
Budding Yeast ◽  
S Phase ◽  
Force Balance ◽  
Replication Forks ◽  
Origin Firing ◽  
Spindle Structure ◽  
Kinetochore Function ◽  
S Phase Checkpoint

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

scholarly journals Stochasticity Triggers Activation of the S-phase Checkpoint Pathway in Budding Yeast

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peijie Zhou ◽  
Xin Gao ◽  
Xiaoli Li ◽  
Linxi Li ◽  
Caoyuan Niu ◽  
...  
Keyword(s):  
Budding Yeast ◽  
S Phase ◽  
Checkpoint Pathway ◽  
S Phase Checkpoint

scholarly journals The Function and Regulation of Budding Yeast Swe1 in Response to Interrupted DNA Synthesis

2006 ◽  
Vol 17 (6) ◽  
pp. 2746-2756 ◽  
Author(s):  
Hong Liu ◽  
Yanchang Wang
Keyword(s):  
Dna Synthesis ◽  
Budding Yeast ◽  
S Phase ◽  
Negative Regulator ◽  
Basic Unit ◽  
Origin Firing ◽  
Protein Levels ◽  
Late Origin ◽  
S Phase Checkpoint

Periodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. Δhsl1, Δhsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1–mediated mitotic activities, but has little effects on Clb5-Cdk1–associated S-phase progression.

scholarly journals Checkpoint phosphorylation sites on budding yeast Rif1 protect nascent DNA from degradation by Sgs1-Dna2

2020 ◽  
Author(s):  
Chandre Monerawela ◽  
Shin-ichiro Hiraga ◽  
Anne D. Donaldson
Keyword(s):  
Protein Phosphatase ◽  
Genome Instability ◽  
Budding Yeast ◽  
S Phase ◽  
Protein Phosphatase 1 ◽  
Phosphorylation Sites ◽  
Replication Forks ◽  
Dna Protection ◽  
S Phase Checkpoint

AbstractIn budding yeast the Rif1 protein is important for protecting nascent DNA at blocked replication forks, but the mechanism has been unclear. Here we show that budding yeast Rif1 must interact with Protein Phosphatase 1 to protect nascent DNA. In the absence of Rif1, removal of either Dna2 or Sgs1 prevents nascent DNA degradation, implying that Rif1 protects nascent DNA by targeting Protein Phosphatase 1 to oppose degradation by the Sgs1-Dna2 nuclease-helicase complex. This functional role for Rif1 is conserved from yeast to human cells. Yeast Rif1 was previously identified as a target of phosphorylation by the Tel1/Mec1 checkpoint kinases, but the importance of this phosphorylation has been unclear. We find that nascent DNA protection depends on a cluster of Tel1/Mec1 consensus phosphorylation sites in the Rif1 protein sequence, indicating that the intra-S phase checkpoint acts to protect nascent DNA through Rif1 phosphorylation. Our observations uncover the pathway by which budding yeast Rif1 stabilises newly synthesised DNA, highlighting the crucial role Rif1 plays in maintaining genome stability from lower eukaryotes to humans.Author summaryGenome instability is a leading factor contributing to cancer. Maintaining efficient error-free replication of the genome is key to preventing genome instability. During DNA replication, replication forks can be stalled by external and intrinsic obstacles, leading to processing of nascent DNA ends to enable replication restart. However, the nascent DNA must be protected from excessive processing to prevent terminal fork arrest, which could potentially lead to more serious consequences including failure to replicate some genome sequences. Using a nascent DNA protection assay we have investigated the role of the budding yeast Rif1 protein at blocked replication forks. We find that Rif1 protects nascent DNA through a mechanism that appears conserved from yeast to humans. We show that budding yeast Rif1 protects nascent DNA by targeting Protein Phosphatase 1 activity to prevent degradation of nascent DNA by the Sgs1-Dna2 helicase-nuclease complex. Furthermore, we find that Rif1 phosphorylation by the checkpoint pathway during replication stress is crucial for this function. Our results indicate that the S phase checkpoint machinery acts by phosphorylating Rif1 to protect nascent DNA, providing important clues concerning the conserved role of Rif1 in regulating events when replication is challenged.

scholarly journals The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

2005 ◽  
Vol 168 (7) ◽  
pp. 999-1012 ◽  
Author(s):  
Jeff Bachant ◽  
Shannon R. Jessen ◽  
Sarah E. Kavanaugh ◽  
Candida S. Fielding
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
S Phase ◽  
Origin Of Replication ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Hydroxyurea Treatment ◽  
Chromatid Cohesion ◽  
S Phase Checkpoint

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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