Saccharomyces cerevisiae W303
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ABSTRACT:
PROVIDER: PRJNA170884 | ENA |
REPOSITORIES: ENA
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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
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
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
Project description:TrAEL-seq was performed on hydroxyurea-blocked and then released yeast cells to track replication fork stalling and replication fork restart, in wild-type and replisome mutant strains.
2023-07-23 | GSE235881 | GEO
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
Project description:The Bloom syndrome helicase BLM interacts with topoisomerase IIIa (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover products during homologous recombination (HR). BLM also promotes DNA-end resection and stalled replication fork restart. However, it is still unclear how the activities of the BTR complex are regulated in cells. Here, we identify multiple highly conserved short linear peptide motifs within the BTR complex that interact cooperatively with RPA. Furthermore, we demonstrate that RPA-binding is essential for stable recruitment of BLM to DNA damage sites and for stalled fork restart, but not for its roles in HR. Disruption of RPA-binding thus constitutes a novel separation-of-function mutation of the BTR complex, and suggest a model in which BTR contains the intrinsic ability to measure levels of RPA-ssDNA at replication forks, which controls its recruitment and activation in response to replication stress.
2021-01-05 | PXD018322 | Pride
Project description:The fidelity of DNA replication is governed by a network of DNA repair mechanisms. Defects in replication fork protection can fuel genome instability in cancer, while also creating cancer-specific vulnerabilities. Here, we provide a comprehensive proteomic characterization of the replication fork response to topoisomerase 1 inhibition, a widely used mainstay chemotherapy. We reveal profound changes in the fork proteome and chromatin environment in response to seDSBs and topological stress, and classify fork repair factors according to their specificity for broken and stalled forks. These distinct fork responses also oppositely affect nucleosome occupancy and nuclear membrane interactions. ATM inhibition dramatically rewired the fork breakage response, revealing that ATM promotes HR-dependent fork restart by concomitantly promoting DNA-end resection and suppressing DSB associated protein ubiquitination and NHEJ. Together, this collection of damaged fork proteomes provides an ample resource to understand fork repair mechanisms and identify druggable targets and novel cancer vulnerabilities.
2021-01-15 | PXD018092 | Pride
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
Project description:DNA replication fidelity is essential for maintaining genetic stability. Forks arrested at replication fork barriers can be stabilised by the intra-S phase checkpoint, subsequently being rescued by a converging fork, or resuming when the barrier is removed. However, some arrested forks cannot be stabilised and fork convergence cannot rescue in all situations. Thus, cells have developed homologous recombination-dependent mechanisms to restart persistently inactive forks. To understand the dynamics of HR-restart, we visualized in vivo replication dynamics at an S. pombe replication barrier, RTS1, using polymerase usage sequencing and model replication dynamics by Monte Carlo simulation. We confirm that HR-restarted forks synthesise both strands with Pol d and that Pol a is not used significantly on either strand: the lagging strand template remains as a gap that is filled in later. We further demonstrate that HR-restarted forks progress for >30 kb kilobases without maturing to a d/e configuration and can progress through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, demonstrating the stability of the 3' single strand in the context of increased resection.
2020-07-03 | GSE153731 | GEO
Project description:R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co- transcriptionally in the opposite orientation to replication fork progression. Recent studies have shown that replication forks stalled by co-transcription R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds RNA:DNA hybrids in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4 pathway for resolution of R-loop-mediated transcription- replication conflicts, which may be involved in R-loop unwinding.
2022-11-02 | PXD034331 | Pride