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PROVIDER: PRJEB50302 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We now report a sequencing method for the measurement of replication fork movement on single molecules by Detecting Nucleotide Analogue signal currents on extremely long nanopore traces (D‑NAscent). Using this method, we detect BrdU incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse labelling period. Under conditions of limiting BrdU concentration, D-NAscent detects the differences in BrdU incorporation frequency across individual molecules to reveal the location of active replication origins, fork direction, termination sites, and fork pausing/stalling events. We used sequencing reads of 20-160 kb, to generate the first whole genome single-molecule map of DNA replication dynamics and discover a new class of low frequency stochastic origins in budding yeast.
2019-03-29 | GSE121941 | GEO
Project description:Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).
2022-02-18 | GSE196761 | GEO
Project description:Every nucleosome across the genome must be disrupted and reformed when the replication fork passes, but how chromatin organization is re-established following replication is unknown. To address this problem, we have developed Mapping In vivo Nascent Chromatin with EdU and sequencing (MINCE-seq) to characterize the genome-wide location of nucleosomes and other chromatin proteins behind replication forks at high temporal and spatial resolution. We find that the characteristic chromatin landscape at Drosophila promoters and enhancers is lost upon replication. The most conspicuous changes are at promoters that have high levels of RNA polymerase II (RNAPII) stalling and DNA accessibility and show specific enrichment for the BRM remodeler. Enhancer chromatin is also disrupted during replication, suggesting a role for transcription factor (TF) competition in nucleosome re-establishment. Thus, the characteristic nucleosome landscape emerges from a uniformly packaged genome by the action of TFs, RNAPII and remodelers minutes after replication fork passage.
2016-04-07 | GSE76120 | GEO
Project description:The higher-order structure of newly replicated (i.e. ‘nascent’) chromatin fibers remains poorly-resolved, limiting our understanding of how epigenomes are maintained across cell divisions. To address this, we present Replication-Aware Single-molecule Accessibility Mapping (RASAM), a long-read sequencing method that nondestructively measures genome-wide replication-status and protein-DNA interactions simultaneously on intact chromatin templates. We report that individual human and mouse nascent chromatin fibers are ‘hyperaccessible’ compared to steady-state chromatin. This hyperaccessibility occurs at two, coupled length-scales: first, individual nucleosome core particles on nascent DNA exist as a mixture of partially-unwrapped nucleosomes and other subnucleosomal species; second, newly-replicated chromatin fibers are significantly enriched for irregularly-spaced nucleosomes on individual DNA molecules. Focusing on specific cis-regulatory elements (e.g. transcription factor binding sites; active transcription start sites [TSSs]), we discover unique modes by which nascent chromatin hyperaccessibility is resolved at the single-molecule level: at CCCTC-binding factor (CTCF) binding sites, CTCF and nascent nucleosomes compete for motifs on nascent chromatin fibers, resulting in quantitatively-reduced CTCF occupancy and motif accessibility post-replication; at active TSSs, high levels of steady-state chromatin accessibility are preserved, implying that nucleosome free regions (NFRs) are rapidly re-established behind the fork. Our study introduces a new paradigm for studying higher-order chromatin fiber organization behind the replication fork. More broadly, we uncover a unique organization of newly replicated chromatin that must be reset by active processes, providing a substrate for epigenetic reprogramming.
2024-11-12 | GSE245558 | GEO
Project description:Mapping the root systems of individual trees in a natural community using genotyping-by-sequencing
| PRJEB57572 | ENA
Project description:Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when HR is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 is required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and importantly, that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.
2023-10-24 | PXD035661 | Pride
Project description:Methylation based plasmid binning using nanopore sequencing
| PRJEB54092 | ENA
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:We have established a novel sequencing approach to characterise usage of replicative DNA polymerases in S. pombe. This approach allows us to determine the roles of DNA polymerase delta and epsilon in lagging strand and leading strand DNA synthesis, respectively in genome-wide scale. Utilising the dataset of usage of these polymerases, we also successfully identified DNA replication initiation sites at high resolution. Furthermore, our informatics analysis establishes genome-wide datasets of fork direction rates, replication timing and the probability of replication termination.
2015-02-03 | GSE62108 | GEO