Cut1/separase-dependent roles of multiple phosphorylation of fission yeast cohesin subunit Rad21 in post-replicative damage repair and mitosis

AbstractMutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21 and STAG2 and screened for synthetic lethality with 3,009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunit rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin mutant cancers.

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The Fission Yeast Rad32 (Mre11)-Rad50-Nbs1 Complex Is Required for the S-Phase DNA Damage Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.23.18.6564-6573.2003 ◽

2003 ◽

Vol 23 (18) ◽

pp. 6564-6573 ◽

Cited By ~ 53

Author(s):

Charly Chahwan ◽

Toru M. Nakamura ◽

Sasirekha Sivakumar ◽

Paul Russell ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Chromosome Instability ◽

S Phase ◽

Comparative Genomic ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Damage Repair ◽

Genomic Approach ◽

Telomere Regulation

ABSTRACT Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G2 DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.

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Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

Scientific Reports ◽

10.1038/srep44257 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 6

Author(s):

Charalampos Rallis ◽

StJohn Townsend ◽

Jürg Bähler

Keyword(s):

Fission Yeast ◽

Double Mutant ◽

Genetic Interaction ◽

Genetic Network ◽

Cellular Growth ◽

Regulatory Subunit ◽

Triple Mutant ◽

Network Information ◽

Damage Repair ◽

Dependent Processes

Abstract The Target of Rapamycin (TOR) signalling network plays important roles in aging and disease. The AMP-activated protein kinase (AMPK) and the Gsk3 kinase inhibit TOR during stress. We performed genetic interaction screens using synthetic genetic arrays (SGA) with gsk3 and amk2 as query mutants, the latter encoding the regulatory subunit of AMPK. We identified 69 negative and 82 positive common genetic interactors, with functions related to cellular growth and stress. The 120 gsk3-specific negative interactors included genes functioning in translation and ribosomes. The 215 amk2-specific negative interactors included genes functioning in chromatin silencing and DNA damage repair. Both amk2- and gsk3-specific interactors were enriched in phenotype categories related to abnormal cell size and shape. We also performed SGA screen with the amk2 gsk3 double mutant as a query. Mutants sensitive to 5-fluorouracil, an anticancer drug are under-represented within the 305 positive interactors specific for the amk2 gsk3 query. The triple-mutant SGA screen showed higher number of negative interactions than the double mutant SGA screens and uncovered additional genetic network information. These results reveal common and specialized roles of AMPK and Gsk3 in mediating TOR-dependent processes, indicating that AMPK and Gsk3 act in parallel to inhibit TOR function in fission yeast.

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Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks

Life Science Alliance ◽

10.26508/lsa.201900433 ◽

2019 ◽

Vol 2 (5) ◽

pp. e201900433 ◽

Cited By ~ 2

Author(s):

Anissia Ait-Saada ◽

Olga Khorosjutina ◽

Jiang Chen ◽

Karol Kramarz ◽

Vladimir Maksimov ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Chromatin Remodeling ◽

Replication Fork ◽

Strand Break ◽

Replication Forks ◽

Damage Repair ◽

Single Strand Annealing ◽

Strand Annealing

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

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Fission yeast Uve1 and Apn2 function in distinct oxidative damage repair pathways in vivo

DNA Repair ◽

10.1016/j.dnarep.2003.08.005 ◽

2003 ◽

Vol 2 (11) ◽

pp. 1253-1267 ◽

Cited By ~ 21

Author(s):

J.Lee A Fraser ◽

Erin Neill ◽

Scott Davey

Keyword(s):

Oxidative Damage ◽

Fission Yeast ◽

Damage Repair ◽

Repair Pathways

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Cohesin-independent segregation of sister chromatids in budding yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e11-08-0696 ◽

2012 ◽

Vol 23 (4) ◽

pp. 729-739 ◽

Cited By ~ 48

Author(s):

Vincent Guacci ◽

Douglas Koshland

Keyword(s):

Mitotic Spindle ◽

Budding Yeast ◽

Alternative Mechanism ◽

Yeast Viability ◽

Sister Chromatids ◽

Damage Repair ◽

Independent Segregation ◽

Independent Mechanism ◽

Cohesin Subunit

Cohesin generates cohesion between sister chromatids, which enables chromosomes to form bipolar attachments to the mitotic spindle and segregate. Cohesin also functions in chromosome condensation, transcriptional regulation, and DNA damage repair. Here we analyze the role of acetylation in modulating cohesin functions and how it affects budding yeast viability. Previous studies show that cohesion establishment requires Eco1p-mediated acetylation of the cohesin subunit Smc3p at residue K113. Smc3p acetylation was proposed to promote establishment by merely relieving Wpl1p inhibition because deletion of WPL1 bypasses the lethality of an ECO1 deletion (eco1Δ wpl1Δ). We find that little, if any, cohesion is established in eco1Δ wpl1Δ cells, indicating that Eco1p performs a function beyond antagonizing Wpl1p. Cohesion also fails to be established when SMC3 acetyl-mimics (K113Q or K112R,K113Q) are the sole functional SMC3s in cells. These results suggest that Smc3p acetylation levels affect establishment. It is remarkable that, despite their severe cohesion defect, eco1Δ wpl1Δ and smc3-K112R,K113Q strains are viable because a cohesin-independent mechanism enables bipolar attachment and segregation. This alternative mechanism is insufficient for smc3-K113Q strain viability. Smc3-K113Q is defective for condensation, whereas eco1Δ wpl1Δ and smc3-K112R,K113Q strains are competent for condensation. We suggest that Smc3p acetylation and Wpl1p antagonistically regulate cohesin's essential role in condensation.

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Meiotic cohesin complexes are essential for the formation of the axial element in mice

The Journal of Cell Biology ◽

10.1083/jcb.201201100 ◽

2012 ◽

Vol 197 (7) ◽

pp. 877-885 ◽

Cited By ~ 66

Author(s):

Elena Llano ◽

Yurema Herrán ◽

Ignacio García-Tuñón ◽

Cristina Gutiérrez-Caballero ◽

Enrique de Álava ◽

...

Keyword(s):

Dna Damage ◽

Synaptonemal Complex ◽

Chromosome Segregation ◽

Protein Complex ◽

Essential Role ◽

Strand Break ◽

Double Strand Break Repair ◽

Damage Repair ◽

Cohesin Subunit ◽

Axial Element

Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.

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Effects of the microtubule nucleator Mto1 on chromosomal movement, DNA repair, and sister chromatid cohesion in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e19-05-0301 ◽

2019 ◽

Vol 30 (21) ◽

pp. 2695-2708 ◽

Cited By ~ 3

Author(s):

Jacob Zhurinsky ◽

Silvia Salas-Pino ◽

Ana B. Iglesias-Romero ◽

Antonio Torres-Mendez ◽

Benjamin Knapp ◽

...

Keyword(s):

Dna Repair ◽

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cytoplasmic Factor ◽

Chromosomal Segregation ◽

Sister Chromatids ◽

Chromatid Cohesion ◽

Cohesin Subunit

Although the function of microtubules (MTs) in chromosomal segregation during mitosis is well characterized, much less is known about the role of MTs in chromosomal functions during interphase. In the fission yeast Schizosaccharomyces pombe, dynamic cytoplasmic MT bundles move chromosomes in an oscillatory manner during interphase via linkages through the nuclear envelope (NE) at the spindle pole body (SPB) and other sites. Mto1 is a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus. Here, we test the function of these cytoplasmic MTs and Mto1 on DNA repair and recombination during interphase. We find that mto1Δ cells exhibit defects in DNA repair and homologous recombination (HR) and abnormal DNA repair factory dynamics. In these cells, sister chromatids are not properly paired, and binding of Rad21 cohesin subunit along chromosomal arms is reduced. Our findings suggest a model in which cytoplasmic MTs and Mto1 facilitate efficient DNA repair and HR by promoting dynamic chromosomal organization and cohesion in the nucleus.

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Caffeine-Mediated Override of Checkpoint Controls: A Requirement for rhp6 (Schizosaccharomyces pombe)

Genetics ◽

10.1093/genetics/152.1.61 ◽

1999 ◽

Vol 152 (1) ◽

pp. 61-71

Author(s):

Roy Rowley ◽

Jun Zhang

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Fission Yeast ◽

Cell Cycle Progression ◽

Synthetic Lethality ◽

Temperature Sensitive ◽

Checkpoint Control ◽

Control Activity ◽

Damage Repair ◽

Cell Progression

Abstract Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are arrested or delayed in interphase through the action of checkpoint controls. If the arrested cell is exposed to caffeine, relatively normal cell cycle progression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viability. To address the mechanism of caffeine’s action on cell progression, fission yeast mutants that take up caffeine but are not sensitized to hydroxyurea (HU) by caffeine were selected. Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene. Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal checkpoint responses to HU, radiation, or inactivation of DNA ligase. However, the addition of caffeine does not override the associated cell cycle blocks. Both point and deletion mutations show synthetic lethality at room temperature with temperature-sensitive mutations in cyclin B (cdc13-117) or the phosphatase cdc25 (cdc25-22). These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, promotes entry to mitosis in response to caffeine treatment.

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Schizosaccharomyces pombe Assays to Study Mitotic Recombination Outcomes

Genes ◽

10.3390/genes11010079 ◽

2020 ◽

Vol 11 (1) ◽

pp. 79

Author(s):

Hannah M. Hylton ◽

Bailey E. Lucas ◽

Ruben C. Petreaca

Keyword(s):

Dna Damage ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Molecular Mechanisms ◽

Mitotic Recombination ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Repair

The fission yeast—Schizosaccharomyces pombe—has emerged as a powerful tractable system for studying DNA damage repair. Over the last few decades, several powerful in vivo genetic assays have been developed to study outcomes of mitotic recombination, the major repair mechanism of DNA double strand breaks and stalled or collapsed DNA replication forks. These assays have significantly increased our understanding of the molecular mechanisms underlying the DNA damage response pathways. Here, we review the assays that have been developed in fission yeast to study mitotic recombination.

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