SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks.
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ABSTRACT:
PROVIDER: PRJNA1037365 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26752327_1.fastq.gz | Fastqsanger.gz | |||
| SRR26752327_2.fastq.gz | Fastqsanger.gz | |||
| SRR26752328_1.fastq.gz | Fastqsanger.gz | |||
| SRR26752328_2.fastq.gz | Fastqsanger.gz | |||
| SRR26752329_1.fastq.gz | Fastqsanger.gz |
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Project description:Nuclear pores complexes (NPCs) are genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for DNA repair. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the speed of restarted DNA polymerase. In contrast to Ulp1, this last function occurs independently of SUMO chains formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes the mechanisms by which the NPC environment ensures optimal RDR.
2023-11-13 | GSE247371 | GEO
Project description:Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.
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Project description:Yeast Sen1Senataxin is a RNA/DNA helicase that preserves replication forks across RNA Polymerase II-transcribed genes by counteracting RNA:DNA hybrids accumulation. We show that in Sen1-depleted cells early forks clashing head-on with transcription halt, and impair progression of sister forks within the same replicon. Unsolved replication-transcription collisions trigger the local firing of dormant origins that rescue arrested forks. In sen1 mutants the MRX and Mrc1/Ctf4-complexes protect those forks clashing with transcription by preventing genotoxic fork-resection events mediated by the Exo1 nuclease. Hence, sister forks within the same replicon remain coupled when one of the two forks halts. This is different when forks encounter double strand breaks. Moreover, the local firing of dormant origins is not prevented by checkpoint activation but depends on delayed adjacent forks. Furthermore, a productive head-on clash between replication and transcription requires the tuning of origin firing and coordination between Sen1, the MRX and Mrc1/Ctf4-complexes and Exo1.
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Project description:The AAA+ ATPase VCP regulates the extraction of SUMO and/or ubiquitin modified factors from chromatin. We previously showed that active DNA synthesis is associated with a SUMO-high and ubiquitin-low environment that is maintained by the deubiquitylase USP7. Here we unveil a functional cooperation between USP7 and VCP in the control of DNA replication conserved from Caenorhabditis elegans to mammals. The function of VCP during DNA replication is mediated by its cofactor FAF1, which facilitates the extraction of SUMOylated proteins that accumulate on chromatin upon USP7 inhibition. Supporting this cooperative role, the inactivation of the orthologues of USP7 and FAF1 is synthetic lethal in C. elegans, and USP7 and FAF1 inhibitors display synergistic toxicity. Together, these results reveal a coordinated activity of USP7 and VCPFAF1 in limiting SUMOylation at active replication forks and promoting the extraction and degradation of ubiquitylated proteins.
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Project description:Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences.
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Project description:Post-translational protein modification by the small ubiquitin-related modifier (SUMO) protein regulates numerous cellular pathways, including transcription, cell division and genome maintenance. The SUMO protease Ulp2 modulates many of these SUMO-dependent processes in budding yeast. To investigate changes to the transcriptome of cells lacking Ulp2, whole-genome RNA sequencing (RNA-seq) was employed. Unexpectedly, ulp2Δ cells display a general two-fold increase in transcript levels across two particular chromosomes, Chromosome I (ChrI) and Chromosome XII (ChrXII). Quantification of relative DNA levels showed that ChrI and ChrXII are present at twice their normal copy number in ulp2Δ cells. This double disomy occurs in mutants in multiple genetic backgrounds and does not require passage through meiosis. An abnormal number of chromosomes in a cell, termed aneuploidy, is usually deleterious. However, development of specific aneuploidies allows rapid adaptation to cellular stresses in yeast and other species, and aneuploidy characterizes most tumors. Extra copies of ChrI and ChrXII appear quickly following loss of active Ulp2, suggesting aneuploidy is an adaptive mechanism in cells lacking the functional SUMO protease. The specific aneuploidy in ulp2Δ cells highlights a unique example of a homogeneous, multi-chromosome aneuploidy in response to mutation of a single gene.
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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.
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Project description:SUMOylation, a protein post-translational modification present in all eukaryotes, involves the covalent attachment of SUMO (small ubiquitin-like modifier) to target proteins, modulating their function, localization or stability. One of the least understood features of SUMOylation is formation of polymeric chains and their impact on DNA metabolism. Here, using Schizosaccharomyces pombe,we demonstrated that cells devoid of SUMO chains exhibit elevated spontaneous replication stress and DNA damage. We found that SUMO chains promoted rDNA stability,controlled proper centromeric organization, and in their absence recombination factors accessed more frequently both of those loci. We directly showed increased fork stalling at rDNA and measured higher recombination rates at centromeres upon SUMO chains deficiency. Moreover, we successfully established the split-SUMO-ID proteomics approach in fission yeast to identify SUMO- and Rad52-dependent interactome. Our results suggest that SUMO chains maintain the stability of difficult-to-replicate loci by limiting the access of recombination factors to stalled replication forks.
2024-09-15 | GSE276805 | GEO
Project description:Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage-bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage-bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domains-containing Saccharomyces cerevisiae protein, Esc2, facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause sub-optimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress, while globally prevented at undamaged replicating chromosomes.
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