Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA120567 | ENA |
REPOSITORIES: ENA
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Project description:[original title] Chromosome replication initiates at multiple replicons and terminates when forks converge. In Escherichia coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S-phase and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes replication fork barriers, Rrm3 and Top2 coordinate replication fork progression and fusion at termination regions thus counteracting abnormal genomic transitions. Signal tracks in BED format suitable for visualization on the UCSC genome browser can be found at http://bio.ifom-ieo-campus.it/supplementary/Fachinetti_et_al_MOLCELL_2010
2010-08-28 | GSE19061 | GEO
Project description:Top1 and Top2-mediated replication fork integrity
| PRJNA101273 | ENA
Project description:Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through inhibition of pause release, we determine that pause-release rates are highly variable and most promoter-proximal paused RNA Polymerase II molecules prematurely terminate (~80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with induction of promoter-proximal termination under hyperosmotic stress but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.
2021-09-13 | GSE166202 | GEO
Project description:Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress induced by loss or inhibition of topoisomerase IIα (TOP2α). Plasmid DNA was replicated in mock- or TOP2α-depleted Xenopus egg extracts as previously described (Heintzman et al. 2019). In parallel, replication was performed in the presence of the TOP2 inhibitor ICRF-193 (‘TOP2-i’) as an alternate means of preventing TOP2 activity (Heintzman et al. 2019). Chromatinized plasmid DNA was recovered 18 minutes after the onset of DNA synthesis, when most forks have normally merged but are stalled when TOP2 activity is prevented (Heintzman et al. 2019). Chromatin-bound proteins were recovered (Larsen et al. 2019) then analyzed by chromatin mass spectrometry and quantified by label free quantification.
2023-02-21 | PXD031170 | Pride
Project description:Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetic, genomic and imaging approaches we found that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs, Sen1 at telomeres. rrm3 and sen1 are synthetic lethal, fail to terminate replication exhibiting lagging chromosomes and fragility at TERs and telomeres. sen1 rrm3 build up RNA-DNA hybrids at TERs, sen1 accumulates RNPII at TERs and telomeres. Double mutants exhibit X-shaped gapped or reversed converging forks. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of fork-associated Top1 and Top2 with those of gene loop-associated Top1 and Top2 by preventing DNA and RNA polymerases slowing down when forks encounter transcription head-on or codirectionally, respectively. Hence Rrm3 and Sen1 are essential to generate permissive topological conditions for replication termination.
2022-10-11 | GSE214925 | GEO
Project description:Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetic, genomic and imaging approaches we found that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs, Sen1 at telomeres. rrm3 and sen1 are synthetic lethal, fail to terminate replication exhibiting lagging chromosomes and fragility at TERs and telomeres. sen1 rrm3 build up RNA-DNA hybrids at TERs, sen1 accumulates RNPII at TERs and telomeres. Double mutants exhibit X-shaped gapped or reversed converging forks. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of fork-associated Top1 and Top2 with those of gene loop-associated Top1 and Top2 by preventing DNA and RNA polymerases slowing down when forks encounter transcription head-on or codirectionally, respectively. Hence Rrm3 and Sen1 are essential to generate permissive topological conditions for replication termination.
2022-10-11 | GSE214908 | GEO
Project description:RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.
2023-09-11 | GSE218927 | GEO
Project description:RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.
2023-09-11 | GSE218924 | GEO
Project description:Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated Top2 contribution in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes without influencing their transcription. The Top2-bound loci exhibit low nucleosome density and accumulate yH2A when Top2 is defective. These intergenic loci associate with the HMG-protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate a S-phasen architectural pathway controlling replicon dynamics when forks encounter transcriptionto preserve genome integrity. Signal tracks in BED format suitable for visualization on the UCSC genome browser can be found at http://bio.ifom-ieo-campus.it/supplementary/Bermejo_et_al_CELL_2009
2009-09-05 | GSE16258 | GEO
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis
2007-08-01 | GSE8305 | GEO