Saccharomyces cerevisiae
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PROVIDER: PRJNA292617 | ENA |
REPOSITORIES: ENA
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Project description:Saccharomyces cerevisiae encodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging- strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes; consistent with this observation, we find that head-on collisions between tRNA transcription and replisome progression are under-represented in the S. cerevisiae genome. Further, we demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.
2016-10-17 | GSE71973 | GEO
Project description:Replication stress activates the Mec1ATR and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1.
2015-09-24 | GSE68214 | GEO
Project description:Replication stress activates the Mec1ATR and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1. BrdU incorporation profiles by ssDNA-BrdU IP on chip have been generated as described (Katou et al., 2003). Protein binding profiles by ChIP-chip analysis were generated as described (Bermejo et al., 2009). Labeled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
2015-09-24 | E-GEOD-68214 | biostudies-arrayexpress
Project description:Background G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. Results We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites. Conclusions In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.
2014-12-03 | GSE59178 | GEO
Project description:tRNA genes (tDNAs) are widely studied sites of replication-fork arrest and genome instability in the budding yeast Saccharomyces cerevisiae. tDNAs are extremely highly transcribed, and serve as constitutive condensin binding sites. Although tRNA transcription by RNA polymerase III (RNAPIII) has previously been identified as stimulating replication-fork arrest at these loci, the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of transcription factor binding, transcription, topoisomerase activity, and condensin-mediated clustering to replication-fork arrest at tDNAs in yeast. We show that a polar block to replication is maintained at tDNAs even when tRNA transcription is abolished by depletion of one or more subunits of RNAPIII. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication in the same background. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork mobility past tDNAs is unaffected by the global depletion of condensin from the nucleus, but can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.
2019-11-02 | GSE139860 | GEO
Project description:Rad53-mediated regulation of Rrm3 and Pif1 DNA helicases contributes to prevention of aberrant fork transitions under replication stress.
| PRJNA282034 | ENA
Project description:Accumulative studies indicate that DNA maintenance methylation by DNMT1 is initiated by binding of UHRF1 to replication fork. However, how UHRF1 gains access to chromatin in S phase is poorly understood. Here we report that LSH, a SNF2 family chromatin remodeler, facilitates DNA methylation in somatic cells primarily by promoting DNA methylation by DNMT1. We show that knockout of LSH in various somatic cells resulted in substantial reduction of DNA methylation, whereas knockout of DNMT3A and DNMT3B only moderately reduced the level of DNA methylation. Consistent with a role in maintenance methylation, genome-wide analysis of DNA methylation revealed a widespread reduction of DNA methylation in all genomic elements in LSH null cells. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association, UHRF1-catalyzed H3 ubiquitination, and subsequent DNMT1 recruitment to replication fork. Notably, UHRF1 also enhances LSH association with replication fork. Thus, our study identifies LSH as an essential factor for maintenance methylation and provides novel insight into how LSH facilitates maintenance methylation.
2020-12-01 | GSE136931 | GEO
Project description:Mammalian DNA replication relies on various DNA helicases and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN: FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks..
2024-08-21 | GSE269605 | GEO
Project description:Replication-fork arrest at tRNA genes in S. cerevisiae does not require tRNA transcription and is facilitated by topoisomerases and Rad18-dependent repair pathways
| PRJNA587158 | ENA
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 • The goal of the experiment Genome-wide localization of Ino80 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 in random culture Distribution of Ino80 in G1 phase Distribution of Ino80 in early S phase • Experimental design ChIP analyses: W303 background cells expressing Myc-tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.
2008-04-10 | E-GEOD-9420 | biostudies-arrayexpress