Chromatin structure governs stalled replication fork processing and cohesin loading
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ABSTRACT:
PROVIDER: PRJNA420042 | ENA |
REPOSITORIES: ENA
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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 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:Replication fork reversal is a key protective mechanism against replication stress in higher eukaryotic cells and occurs via a series of coordinated enzymatic reactions. The Bloom syndrome gene product, BLM, is a member of the highly conserved RecQ helicase family and has been implicated in this process, but its precise regulation and role remain poorly understood. Here, we show that, upon replication stress, the GCFC domain-containing protein TFIP11 competes with the BLM helicase for association with stalled replication forks, thereby facilitates RAD51-mediated stalled fork reversal. Consequently, loss of TFIP11 results in aberrant accumulation of BLM at stalled forks, which in turn compromises RAD51 recruitment, impairs replication stress-induced fork reversal and slowing, hypersensitizes cells to replication stress-inducing agents, and enhances chromosomal instability. These findings reveal a previously unidentified regulatory mechanism that modulates the activities of BLM and RAD51 at stalled forks and thus genome integrity.
2024-05-24 | PXD042222 | Pride
Project description:DNA polymerase epsilon (Pole) carries out leading strand synthesis with high fidelity owing to its exonuclease activity. Pole polymerase and exonuclease activities are in balance, due to partitioning of nascent strands between catalytic sites, so that net end resection occurs when synthesis is impaired. Stalling of chromosomal DNA synthesis activates replication checkpoint kinases, required to preserve the functional integrity of replication forks. We found that Pole is phosphorylated in a Rad53CHK1-dependent manner upon fork stalling, likely to limit Pole-driven nascent strand resection that causes replication fork collapse. In stress conditions Pole phosphorylation occurs on serine 430 of the Pol2 catalytic subunit. A S430 phosphomimic limits strand partitioning and exonucleolytic processivity, while non-phosphorylatable Pol2-S430A bypasses checkpoint regulation causing stalled fork resection and collapse. We propose that checkpoint kinases switch Pole to an exonuclease-safe mode by curbing active site partitioning thus preventing nascent strand resection and stabilizing stalled replication forks.
2021-04-30 | GSE156480 | GEO
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
Project description:Oncogene-induced replication stress constitutes an early obstacle for pre-cancerous cells to
overcome to progress towards malignancy. Fanconi anaemia signalling represents a major
genomic maintenance pathway that is activated in response to replication stress, impinging on
stalled replication fork stability and recovery. Here, we report that FBXL12 ubiquitinates the
central Fanconi anaemia protein FANCD2 at stalled replication forks upon CHK1-mediated
phosphorylation, resulting in FANCD2 degradation. This mechanism is required to promote
efficient and faithful DNA replication under conditions of cyclin E- and drug-induced
replication stress. In the absence of FBXL12, FANCD2 becomes trapped on chromatin leading
to replication stress, excessive DNA damage, and cell death. In human cancers, FBXL12, cyclin
E, and Fanconi anaemia signalling are positively correlated and upregulation or amplification
of FBXL12 is linked to reduced survival in patients with high cyclin E expressing breast
tumours. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and
sensitised breast cancer cells to drug-induced replication stress by WEE1 inhibition.
Collectively, our results indicate that FBXL12 constitutes a vulnerability of cyclin E-
overexpressing cancer cells and represents a novel target for cancer therapy.
2023-05-22 | MSV000092004 | MassIVE
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:DNA replication is frequently perturbed by intrinsic, as well as extrinsic, genotoxic stress. At damaged forks, DNA replication and repair activities require proper coordination to maintain genome integrity. We show here that PARI antirecombinase plays an essential role in modulating the initial response to replication stress in mice. PARI is functionally dormant at replisomes during normal replication, but upon replication stress, it enhances nascent-strand shortening that is regulated by RAD51 and MRE11. PARI then promotes double-strand break induction, followed by new origin firing instead of replication restart. Such PARI function is apparently obstructive to replication but is nonetheless physiologically required for chromosome stability in vivo and ex vivo Of note, Pari-deficient embryonic stem cells exhibit spontaneous chromosome instability, which is attenuated by differentiation induction, suggesting that pluripotent stem cells have a preferential requirement for PARI that acts against endogenous replication stress. PARI is a latent modulator of stalled fork processing, which is required for stable genome inheritance under both endogenous and exogenous replication stress in mice.
| S-EPMC5686578 | biostudies-literature
Project description:The RSC chromatin remodeling complex recruits the cohesin loader Scc2-Scc4 by a direct protein interaction. Nucleosome-free DNA is the substrate for the cohesin loading reaction.
2019-03-19 | GSE117881 | GEO