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Cohesin association to replication sites depends on Rad50 and promotes fork restart

ABSTRACT: The cohesin complex holds together newly-replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on H2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50delta mutants and cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a novel function for cohesin in the maintenance of genome integrity during S phase.

ORGANISM(S): Saccharomyces cerevisiae W303 Saccharomyces cerevisiae

PROVIDER: GSE39078 | GEO | 2012/10/01

SECONDARY ACCESSION(S): PRJNA170884

REPOSITORIES: GEO

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Similar Datasets

Cohesin association to replication sites depends on Rad50 and promotes fork restart

Project description:The cohesin complex holds together newly-replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on H2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50delta mutants and cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a novel function for cohesin in the maintenance of genome integrity during S phase. Scc1 ChIP, Rad50 ChIP and BrdU IP in wild type and mutants at different stages of the cell cycle.

2012-10-01 | E-GEOD-39078 | biostudies-arrayexpress

Genome-wide ChIP-chip Rad50 in early S-phase in the presence of hydroxyurea

Project description:The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSB). A mutation in the N-terminal OB-fold of the 70-kD subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end resection. High resolution imaging shows that either the rfa1-t11 or the rad50 mutation lets stalled replication forks collapse, and allows the separation not only of opposing ends, but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA -MRX contacts are intact, we conclude that MRX also serves as a structural lynchpin of sister chromatids at breaks. Rad50 is a member of the Mre11-Rad50-Xrs2 (MRX) complex which is known to associate to replication forks under hydroxyurea-stress condition. Using ChIP-chip, we show that MRX recruitment to replication forks partially rely on Rfa1 (RPA) at the genome-wide level.

2016-10-18 | GSE88816 | GEO

MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Project description:Rad50 is a component of the conserved MRE11-RAD50-NBS1 (MRN) complex, which functions in genome stability and the cell’s ability to deal with stalled DNA replication forks. We identified Rad50 as a factor important for R-loop tolerance and thus mapped DNA:RNA hybrids in Rad50KO cells and compare them to previously reported wild-type and Sgs1KO profiles.

2019-10-31 | E-MTAB-7885 | biostudies-arrayexpress

CST interacts with the cohesin complex and promotes chromosome cohesion

Project description:Sister chromatid cohesion (SCC), the pairing of sister chromatids following DNA replication until mitosis, is established by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. However, how SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress may lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST associates with the cohesin complex. Unexpectedly, we did not find evidence of altered cohesion or mitotic progression in the absence of CST; however, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we hypothesized that CST may be required to maintain or remodel SCC following DNA replication fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.

2021-07-28 | PXD026264 | Pride

Chromatin structure governs stalled replication fork processing and cohesin loading

Project description:The progression of replication forks (RFs) can be challenged by obstacles of endogenous or exogenous origin. Stalled forks need to be readily stabilized and restarted in order to prevent genomic instability. Replication fork restart requires the recruitment of multiple enzymes and involves different DNA transactions. The MRX (Mre11-Rad50-Xrs2) complex plays a central role in this process. It has been implicated in the nucleolytic degradation of nascent DNA and in the loading of cohesin at stalled forks. However, little is known on how these functions are regulated. Here we show that MRX structural features are predominant on the nuclease activity of Mre11 for DNA resection at stalled replication forks. This results raise the question of the mechanisms by which MRX promotes nascent strand resection. At DNA double strand breaks (DSB) MRX promotes the binding of the chromatin remodeler RSC, from which the activity is required for 5’ end resection. Interestingly, MRX mutants exhibit increased nucleosome occupancy at stalled replication forks. This result suggest that a dynamic chromatin structure promoted by MRX could be required for the processing of stalled replication forks. Strinkingly the absence of two histones modifiers Gcn5 and Set1 recapitulates the phenotype of MRX mutants both on chromatin structure and nascent strand resection at stalled forks even though they are dispensable for its recruitment. Since nucleosomes also represent obstacles for the loading of SMC complexes on DNA, it is coherent that we observed that cohesin is no longer recruited at stalled forks in gcn5 and set1, as in MRX mutants. Together our data suggest that the regulation of chromatin structure at stalled replication forks is essential for their processing and to promote genome stability.

2019-10-04 | GSE107420 | GEO

Cohesin and cohesion establishment factors distribution on S.Cerevisiae chromosome VI during the cell cycle

Project description:Two identical sister copies of eukaryotic chromosomes are synthesised during S-phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onwards by the chromosomal cohesin complex. It is thought that replication fork progression is coupled to establishment of sister chromatid cohesion, thus facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4 and Ctf18 are found close to, and Ctf4 travels along chromosomes with replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S-phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment, and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved. Keywords: ChIP on chip

2006-09-02 | GSE5719 | GEO

Cohesin releases DNA through asymmetric ATPase-driven ring opening

Project description:Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesinâs Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover an unexpected functional asymmetry within the heart of cohesinâs highly conserved ABC-like ATPase machinery and show that an activity associated with one of cohesinâs two ATPase sites drives DNA release from cohesin rings. This key mechanism is conserved from yeast to humans. We propose that Eco1 locks cohesin rings around the sister chromatids by counteracting an asymmetric cohesin-associated ATPase activity. Effect of mutations in Smc1 and Smc3 on cohesin loading onto chromosomes

2016-02-18 | E-GEOD-76891 | biostudies-arrayexpress

Genowide-wide analysis of scc2-4 binding.

Project description:The evolutionarily conserved cohesin complex is crucial for holding sister chromatids together from the time of DNA replication until their segregation during the metaphase to anaphase transition. Human diseases associated with with mutations in the cohesin network are termed "cohesinopathies". Scc2 is required for loading cohesin onto DNA prior to DNA replication. Cornelia de Lange syndrome (CdLS), a developmental disorder characterized by growth and intellectual impairment is caused by mutations in Scc2. How mutations in Scc2 gives rise to these developmental defects is currently unknown, as overt defects in chromosome segregation are not observed in CdLS patients. One hypothesis is that, reduced binding of Scc2 causes gene misregulation. To further explore this idea ChIP-seq was performed using an scc2-4 mutant. We observed from the analysis, we observe reduce binding of Scc2 to Pol II transcribed genes (snoRNAs, ribosomal protein genes) and pol II transcribed genes (tRNAs). Studies are currently underway to examine the biological implications of this observation. Examining genome-wide binding of scc2-4 mutant

2015-06-16 | E-GEOD-55357 | biostudies-arrayexpress

The cohesin loader Scc2 regulates key regulatory genes critical for development

Project description:The evolutionarily conserved cohesin complex is crucial for holding sister chromatids together from the time of DNA replication until their segregation during the metaphase to anaphase transition. Human diseases associated with with mutations in the cohesin network are termed "cohesinopathies". Scc2 is required for loading cohesin onto DNA prior to DNA replication. Cornelia de Lange syndrome (CdLS), a developmental disorder characterized by growth and intellectual impairment is caused by mutations in Scc2. How mutations in Scc2 gives rise to these developmental defects is currently unknown, as overt defects in chromosome segregation are not observed in CdLS patients.This has led to the hypothesis that developmental disorders in CdLS patients are a result of dysregulated gene expression. To examine the transcriptional program of Scc2 mutants called scc2-4, RNA sequencing was performed. Analysis of gene expression program shows upregulation of genes involved in ribosome biogenesis and downregulation of genes needed for oxidative phosphorylation. Studies are currently underway to investigate how scc2-4 mutation causes gene misregulation. The answer(s) could provide insight into the molecular etiology of CdLS. Examining gene expression in 3 biological replicates of scc2-4 relative to wt by Ilumina sequencing

2015-06-16 | E-GEOD-55316 | biostudies-arrayexpress

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart

Project description:R-loops represent a major source of replication stress but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest and restart in S. cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate normally their chromosomes under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Indeed, these cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative step in resuming replication. Similar impairments in nascent DNA resection at HU-arrested forks were observed in human cells lacking RNase H2. However, the addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation, fully restored fork resection. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

2023-10-14 | GSE215896 | GEO

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