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A chromatin-associated long non-coding RNA is required for effective replication stress signaling [RNAseq_HUtreatment]

ABSTRACT: Despite the well-characterized protein network involved in replication stress response, several regulatory RNAs have been shown to be involved in this process; however, it has remained elusive the presence of such RNAs on the replication fork, and how they locally act in this critical process. Here, we identified lncREST, a previously uncharacterized lncRNA among a set of chromatin-associated transcripts induced by hydroxyurea. We show that lncREST localizes on stressed replication forks. Specifically, during replication stress, lncREST interacts with NCL in the nucleus, to help engage its interaction with RPA. Consequently, lncREST loss is associated to reduction of NCL-RPA interaction and decreased RPA on chromatin, leading to a defective replication stress signaling. lncREST depleted cells suffer sustained replication fork progression and accumulate un-signaled DNA damage. These findings uncover a structural function of a lncRNA acting as anchoring point for replication proteins foci on sites of DNA replication.

ORGANISM(S): Homo sapiens

PROVIDER: GSE229868 | GEO | 2023/12/18

REPOSITORIES: GEO

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A chromatin-associated long non-coding RNA is required for effective replication stress signaling [RNAseq_cell_cycle]

Project description:Despite the well-characterized protein network involved in replication stress response, several regulatory RNAs have been shown to be involved in this process; however, it has remained elusive the presence of such RNAs on the replication fork, and how they locally act in this critical process. Here, we identified lncREST, a previously uncharacterized lncRNA among a set of chromatin-associated transcripts induced by hydroxyurea. We show that lncREST localizes on stressed replication forks. Specifically, during replication stress, lncREST interacts with NCL in the nucleus, to help engage its interaction with RPA. Consequently, lncREST loss is associated to reduction of NCL-RPA interaction and decreased RPA on chromatin, leading to a defective replication stress signaling. lncREST depleted cells suffer sustained replication fork progression and accumulate un-signaled DNA damage. These findings uncover a structural function of a lncRNA acting as anchoring point for replication proteins foci on sites of DNA replication.

2023-12-18 | GSE229869 | GEO

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.

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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.

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Genome-wide analysis of replication fork locations in hydroxyurea-treated Saccharomyces cerevisiae by TrAEL-seq

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.

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The Bloom Syndrome Complex Senses RPA-Coated Single-Stranded DNA to Restart Stalled Replication Forks

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.

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A regulatory phosphosite on Mec1 controls RNAPII and RNAPIII occupancy during replication stress [copy_number]

Project description:Upon replication stress, the Mec1ATR kinase triggers the downregulation of transcription, reducing the level of RNA polymerase on chromatin to facilitate replication fork progression. We identify a hydroxyurea-induced phosphorylation site at Mec1-S1991 that contributes to the eviction of RNAPII and RNAPIII during replication stress. The non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII with a Rpb3-TAP fusion, suggesting that lethality arises from replication-transcription conflicts. Coincident with a failure to repress gene expression, highly transcribed genes like GAL1 persist at nuclear pores in mec1-S1991A cells. Consistently, we find pore proteins and several components controlling RNAPII and RNAPIII transcription are phosphorylated in a Mec1-dependent manner, suggesting that Mec1-S1991 phosphorylation limits conflicts between replication and either RNA polymerase complex. We further show that Mec1 contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase.

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A regulatory phosphosite on Mec1 controls RNAPII and RNAPIII occupancy during replication stress [ChIP-seq]

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A regulatory phosphosite on Mec1 controls RNAPII and RNAPIII occupancy during replication stress [tDNA]

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Genome-wide fork-collapse sites in MEFs from ATR inhibition

Project description:Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use RPA ChIP-Seq to identify replication-problematic loci (RPLs) across the mammalian genome from ATR inhibition.

2018-10-04 | GSE100790 | GEO

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly

Project description:The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression. S.cerevisiae oligonucleotide microarrays were provided by Affymetrix (S.cerevisiae Tiling 1.0R, P/N 900645). BrdU and proteins ChIP-chip analyses were carried out as described (Fachinetti et al., M Cell, 2010).

2014-09-12 | E-GEOD-61030 | biostudies-arrayexpress

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