Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA148291 | ENA |
REPOSITORIES: ENA
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Project description:Intracellular levels of deoxyribonucleoside triphosphate (dNTP) must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools have recently been associated with increased mutagenesis, genomic instability and tumorigenesis. However, the mechanisms by which low or imbalanced dNTP pools affect DNA replication remain poorly understood. Here, we have modulated the activity of ribonucleotide reductase (RNR), a key enzyme catalyzing a rate-limiting step of dNTP production, to monitor the effect of altered dNTP levels on replication dynamics in budding yeast. We show that dNTP pools are limiting for normal DNA synthesis as upregulation of RNR activity increases replication fork speed. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition from a regular- to a slow-replication mode within minutes after S-phase entry. Interestingly, we found that upregulation of RNR activity delays this transition and that dNTP levels modulate both fork speed and origin usage under replication stress. Moreover, we report that chromosomal instability (CIN) mutants show increased dNTP pools and enhanced DNA synthesis in the presence of HU. Since upregulation of RNR allows forks to progress faster in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.
2012-01-01 | GSE33686 | GEO
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
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.
2014-09-12 | GSE61030 | GEO
Project description:Top1 and Top2-mediated replication fork integrity
| PRJNA101273 | ENA
Project description:Mapping Replication Fork Pause Sites in S. cerevisiae
| PRJNA117157 | ENA
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:Mannose is an anti-cancer sugar that inhibits cell proliferation and enhances chemotherapy. How mannose exerts its anti-cancer activities, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced a global metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphate (dNTP) pools. This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, a pharmacological inhibition of de novo dNTP biosynthesis was sufficient to sensitize the non-engineered cells to cisplatin and inhibit dormant origin firing, suggesting the dNTP loss-induced genome instability as a major mechanism for the anti-cancer activities of mannose.
2023-07-06 | PXD036449 | Pride
Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip
2008-04-10 | GSE9420 | GEO
Project description:In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and the concentrations of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Although it is documented that imbalanced dNTP pool expansion increases mutation frequency in cancer cells, it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here we have investigated how fibroblasts isolated from Aicardi Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell-cycle progression, dynamics and fidelity of DNA replication, efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages than wildtype fibroblasts. To investigate if the lack of SAMHD1 affects DNA replication fidelity we compared de novo mutations in AGS and WT cells by exome next generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic promoting genome instability in non-transformed cells.
2019-12-03 | GSE135652 | GEO
Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.
2018-02-11 | GSE89721 | GEO