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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. Scc1 ChIP, Rad50 ChIP and BrdU IP in wild type and mutants at different stages of the cell cycle.

ORGANISM(S): Saccharomyces cerevisiae W303

SUBMITTER: Philippe Pasero

PROVIDER: E-GEOD-39078 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

2012-10-01 | GSE39078 | GEO

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

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

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

Histone chaperone NAP1 is required for sister chromatid resolution

Project description:The fidelity of chromosome duplication through cell divisions requires timely binding and release of the cohesin. Cohesin is a ring-shaped protein complex linking newly replicated sister chromatids to ensure their appropriate transmission through mitosis. Upon commencement of mitosis cohesin is removed from DNA in two steps: first, from chromosome arms resulting in sister chromatid resolution, and, second, from centromers leading to sister chromatid segregation. As DNA of eukaryotic chromosomes is assembled into chromatin, regulation of sister chromatid cohesion-segregation may involve chromatin modifying machinery, but this link is not well understood. Here we report that H2A-H2B histone chaperone NAP1, a factor, which is primarily implicated in chromatin assembly, is required for cohesin release from mitotic chromosome arms. NAP1 and cohesin protein complex interact directly and share multiple binding sites on chromatin. Depletion of the NAP1 hinders cohesin removal during mitosis resulting in accumulation of unresolved sister chromatids. Thus, in addition to its well established functions in chromatin dynamics, histone chaperone NAP1 coordinates cell cycle dependent cohesin release. These results reveal a novel molecular pathway for sister chromatid resolution and emphasizes a role for histone chaperones in control of eukaryotic genome replication and transmission. Genome-wide NAP1 and Cohesin ChIP-chip profiling in Drosophila S2 cells. The supplementary bed file S2_cohesin_sites.bed contains cohesin binding sites obtained by intersecting the sets of significant ChIP-chip peaks for SA (a cohesin subunit; stromalin) and SMC1.

2012-07-25 | E-GEOD-30938 | biostudies-arrayexpress

Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites [S-phase/M-phase synchronized cells]

Project description:During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation. Examination of TF binding by ChIP-seq in a CRC cell-line treated with non-targeting siRNA, or siRNA specific to the cohesin subunit RAD21.

2013-10-01 | E-GEOD-51290 | biostudies-arrayexpress

Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites [Validation Set]

2013-09-25 | E-GEOD-51142 | biostudies-arrayexpress

Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites

2013-08-15 | E-GEOD-49402 | biostudies-arrayexpress