Double pulse experiments
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB90580 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We have designed a methodology for capture of DNA 3’ ends that allows mapping of resected DNA breaks, stalled replication forks and also normal replication fork progression. This Transferase-activated end ligation or TrAEL-seq method involves ligation of a functionalised linker to DNA 3’ ends followed by fragmentation, purification of adaptor ligated fragments, second adaptor ligation and library amplification. The major advantages of TrAEL-seq compared to other available methods are: i) an ability to map double strand breaks after resection, ii) excellent sensitivity and signal-to-noise in detecting replication fork stalling and iii) ability to map replication fork progression in unsynchronised, unlabelled populations of both yeast and mammalian cells. The samples provided here were selected to demonstrate different aspects of TrAEL-seq activity: the SfiI and dmc1 datasets shows capture of 3’ extended single strand DNA. The other yeast datasets show replication and replication fork stalling information. The RAF and RAF-GAL grown yeast samples show the effect transcriptional induction on replication fork progression. The hESC samples show the capacity to derive replication profiles from mammalian cells.
2020-07-27 | GSE154811 | GEO
Project description:Cytotoxicity of DNA-protein crosslinks (DPCs) is ascribed largely to their ability to block the progression of DNA replication fork. DPCs are frequently occurring in cells, either as a consequence of metabolism or exogenous agents. The mechanism of DPCs removal is not completely understood. Here, we characterize SPRTN (DVC1) as specialised DNA-dependent metalloprotease for DPC removal in humans. SPRTN has an N-terminal metalloprotease domain that cleaves various DNA binding substrate during S-phase progression. SPRTN is a part of replisome and removes DPCs during DNA replication fork progression, thus protecting proliferative cells from DPCs toxicity. Ruijs-Aalfs Syndrome (RJALS) patient cells with monogenic mutations in SPRTN are hypersensitive to DPC-inducing agents due to DPC removal defect and DNA replication fork stalling. We propose a model where SPRTN protease forms specialised DNA-replication coupled DPC removal pathway essential for DNA replication fork progression and genome stability. We conclude RJALS is the first human syndrome linked to this pathway
2017-03-29 | MSV000080743 | MassIVE
Project description:Cytotoxicity of DNA-protein crosslinks (DPCs) is ascribed largely to their ability to block the progression of DNA replication fork. DPCs are frequently occurring in cells, either as a consequence of metabolism or exogenous agents. The mechanism of DPCs removal is not completely understood. Here, we characterize SPRTN (DVC1) as specialised DNA-dependent metalloprotease for DPC removal in humans. SPRTN has an N-terminal metalloprotease domain that cleaves various DNA binding substrate during S-phase progression. SPRTN is a part of replisome and removes DPCs during DNA replication fork progression, thus protecting proliferative cells from DPCs toxicity. Ruijs-Aalfs Syndrome (RJALS) patient cells with monogenic mutations in SPRTN are hypersensitive to DPC-inducing agents due to DPC removal defect and DNA replication fork stalling. We propose a model where SPRTN protease forms specialised DNA-replication coupled DPC removal pathway essential for DNA replication fork progression and genome stability. We conclude RJALS is the first human syndrome linked to this pathway
2016-10-06 | PXD004154 | Pride
Project description:Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays. We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork. This study is detailed in Khodursky AB et al.(2000) Proc Natl Acad Sci U S A 97:9419-24 Keywords: other
2001-07-30 | E-GEOD-8 | biostudies-arrayexpress
Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.
2022-02-22 | PXD031168 | Pride
Project description:Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays. We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork. This study is detailed in Khodursky AB et al.(2000) Proc Natl Acad Sci U S A 97:9419-24 Keywords: other
2001-07-30 | GSE8 | GEO
Project description:Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a direct regulator of replication fork progression suggesting developmental regulation of Rif1 activity.
2018-10-15 | GSE114370 | GEO
Project description:Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair
| PRJNA277726 | ENA
Project description:DNA replication progression can be affected by the presence of physical barriers on the DNA, like the RNA Polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know the overall influence of transcription on DNA replication progression. To characterize what happens at sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints to identify replication fork/stalling hotspots as the replication progresses through the genome. These sites are typically associated with increased DNA damage, overlap with fragile sites and with breakpoints of rearrangements identified in cancers, but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA Polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. We also find instances where transcription activity favors replication progression because reduces histone density, but also that slowing down transcription elongation slows down directly replication progression through genes. This would indicate that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.
2024-05-13 | GSE267038 | GEO
Project description:DNA replication progression can be affected by the presence of physical barriers on the DNA, like RNA Polymerases, leading to replication stress and DNA damage. To characterize what happens at sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase, to identify replication fork/stalling hotspots throughout the genome. These sites are typically associated with increased DNA damage, overlap with fragile sites and with breakpoints of rearrangements identified in cancers, but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA Polymerase II transcription, we found that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. This would support data that indicate that transcription and replication can coexist over the same regions. We found instances where transcription activity by reducing histone density favors replication progression through genes, but also found that slowing down transcription elongation slows down directly replication progression through genes. Importantly, rearrangements found in cancers at transcription-replication collision sites can be detected in non-transformed cells and increased following treatment with ATM and ATR inhibitors. Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability.
2024-05-13 | GSE169619 | GEO