Replication initiation sites in cancer cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE28911 | GEO | 2011/08/02
SECONDARY ACCESSION(S): PRJNA140633
REPOSITORIES: GEO
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Project description:Reads from massively parallel sequencing of RNA primed, short nascent strands from asynchronously growing cancer cells (K562, MCF7). Newly replicated DNA was isolated based on size (400-800 bp) and the presence of a short RNA stretch at the 5' end using lambda exonuclease. Purified nascent strands were analyzed using massively parallel sequencing. Sheared genomic DNA was sequenced as a control.
2011-08-02 | E-GEOD-28911 | biostudies-arrayexpress
Project description:Genome-wide detection of replication origins by sequencing of short nascent strands (SNS) purified from mouse embryonic stem cells (mESCs).
2022-08-24 | GSE131699 | GEO
Project description:Short nascent strands purification coupled to next-generation sequencing allowed us to identify replication origins on human genome in an extensive way, by mapping replication origins in 4 different cell types, IMR-90 fibroblasts, hESC H9 cells, iPSC Th Cl-4 cells and HeLa cells. We demonstrated the existence of a cell type-specific reprogrammable signature of the cell identity revealed by specific efficiencies of conserved origin positions and not by the selection of cell-type specific subsets of origins. 4 different cell types were analyzed. For each cell types, 2 different biological replicates of short nascent strands at replication origins were purified. Each SNS sample was sequencing at least one time.
2012-07-01 | E-GEOD-37757 | biostudies-arrayexpress
Project description:Mammalian chromosome replication starts from distinct sites, but the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. We identified a replication initiation determinant (RepID) protein that binds a subset of replication initiation sites. A large fraction of RepID binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from Rep-ID bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication initiation events. These observations are consistent with a model suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. Nascent strands were purified with the lambda exonuclease methods from HCT116 cells and sequenced. Chromatin from unsyncrhonized untreated cultures of U2OS cells was subjected to ChIP-Seq with antibody directed against RepID/PHIP
2016-06-02 | E-GEOD-80298 | biostudies-arrayexpress
Project description:Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS) suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation unveiling a new hallmark in ORI efficiency regulation in mammalian cells. Two biological replicates of lambda-exonuclease treated short nascent strands (100-600 or 300-800 nt in length) were co-hybridised with genomic DNA from the same cells to tiled genomic array covering 10.1 Mb of the mouse genome (Agilent Technologies)
2010-05-22 | E-GEOD-15082 | biostudies-arrayexpress
Project description:Faithful duplication of DNA is essential for the maintenance of genomic stability in all organisms. DNA synthesis proceeds bi-directionally with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. Herein, we describe a method of enriching and Sequencing of Protein-Associated Nascent strand DNA (eSPAN) to detect whether a protein binds the leading- and lagging-strands of DNA replication forks. We show that Pol-epsilon, PCNA, Cdc45, Mcm6 and Mcm10 preferentially associate with leading strands, whereas Pol-alpha, Pol32, Pol-delta, Rfa1 and Rfc1 associate with lagging strands of hydroxyurea (HU)-stalled replication forks. In contrast, PCNA is enriched at lagging strands of normal replication forks in wild type cells and HU-stalled forks in cells lacking Elg1. These studies demonstrate a strategy to reveal proteins at leading and lagging strands of DNA replication forks, and suggest that the unloading of PCNA from lagging strands of HU-stalled replication forks helps maintain genome integrity.
2014-10-29 | GSE52614 | GEO
Project description:Replication of the eukaryotic genome occurs in the context of chromatin, a nucleoprotein packaging state consisting of repeating nucleosomes. Chromatin is commonly thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture that occur during genomic replication. Here, we sought to directly measure a key aspect of chromatin structure dynamics during replication â how rapidly nucleosome positions are established on the newly-replicated daughter genomes. By isolating newly-synthesized DNA marked with the nucleotide analogue EdU, we characterize nucleosome positions on both daughter genomes of budding yeast during a time course of chromatin maturation. We find that nucleosomes rapidly adopt their mid log positions at highly-transcribed genes, and that this process was impaired upon treatment with the transcription inhibitor thiolutin, consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in the Hir1Î background reveal a role for HIR in nucleosome spacing. Using strand-specific EdU libraries, we characterize nucleosome positions on the leading and lagging strand daughter genomes, uncovering differences in chromatin maturation dynamics between the two daughter genomes at hundreds of genes. Our data define the maturation dynamics of newly-replicated chromatin, and support a role for transcription in sculpting the chromatin template. We have mapped changes in nucleosome positions on newly replicated DNA in a timecourse after genome replication. We have used Micrococcal Nuclease footprinting of cross linked chromatin to determine nucleosome positions and EdU (ethylene deoxy uridine) to mark nascent DNA strands. EdU incorporated into nascent DNA strands was biotinylated with Click chemistry and nascent DNA strand fragments were subsequently isolated using Streptavidin coated magnetic beads.
2016-08-25 | E-GEOD-74090 | biostudies-arrayexpress
Project description:Faithful duplication of DNA is essential for the maintenance of genomic stability in all organisms. DNA synthesis proceeds bi-directionally with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. Herein, we describe a method of enriching and Sequencing of Protein-Associated Nascent strand DNA (eSPAN) to detect whether a protein binds the leading- and lagging-strands of DNA replication forks. We show that Pol-epsilon, PCNA, Cdc45, Mcm6 and Mcm10 preferentially associate with leading strands, whereas Pol-alpha, Pol32, Pol-delta, Rfa1 and Rfc1 associate with lagging strands of hydroxyurea (HU)-stalled replication forks. In contrast, PCNA is enriched at lagging strands of normal replication forks in wild type cells and HU-stalled forks in cells lacking Elg1. These studies demonstrate a strategy to reveal proteins at leading and lagging strands of DNA replication forks, and suggest that the unloading of PCNA from lagging strands of HU-stalled replication forks helps maintain genome integrity. We synchronized yeast cells at G1 and released into early S phase in the presence of BrdU, a nucleotide analog that can be incorporated into newly synthesized DNA strand, and hydroxyurea (HU), a ribonucleotide reductase inhibitor. HU has no effect on initiation of DNA replication at early replication origins, but inhibit late replication firing. In addition, replication forks are stalled due to depletion of dNTPs. We then performed chromatin-immunoprecipitation of 12 proteins of interest following a standard procedure. Protein-bound DNAs were then reverse-crosslinked and double strand DNA was denatured. Nascent DNA was enriched by immunoprecipitation using anti-BrdU antibodies. The recovered ssDNA was first marked with ligation to one oligo at 3M-bM-^@M-^Y end before conversion to dsDNA for library preparation and sequencing. In this way, the directionality of ssDNA and therefore strand information of each sequenced DNA were known. The sequencing tag was mapped to both Watson (red) and Crick (blue) strands of the reference genome. In addition to ChIP-eSPAN, we also performed BrdU-IP and single strand DNA sequence (BrdU-ssSeq) and protein ChIP followed by single-strand DNA sequencing (ChIP-ssSeq) for each corresponding ChIP-eSPAN experiment. We also performed Mcm4 and Mcm6 ChIP-seq using cells synchronized at G1 phase of the cell cycle for identification of replication origins in comparison with published dataset. Some protein ChIP-ssSeq and ChIP-eSPAN experiments were repeated and the data fits well each other. Therefore, we did not repeat all protein ChIP-ssSeq and ChIP-eSPAN experiments.
2014-10-29 | E-GEOD-52614 | biostudies-arrayexpress
Project description:The tolerance pathway allows bypassing deleterious lesions that block replicative DNA polymerases. In eukaryotes tolerance is controlled by ubiquitylation of the DNA replication factor PCNA4. Here, we show that PCNAK164-ubiquitin proteases (PCNA-DUBs) Ubp10 and Ubp12 localise to replication forks. In particular, the main PCNA-DUB, Ubp10, associates primarily to nascent lagging strands. We also found that cells deficient for PCNA deubiquitylation show both increased interaction of TLS polymerase ζ-associated Rev1 with lagging strands and Rad52-dependent template switching events. This evidence reveals a fork-coupled layer of DDT regulation allowing strand-specific pathway choices.
2019-10-29 | GSE90157 | GEO
Project description:Because of the lack of information, regulation of DNA replication initiation in mammals is still poorly understood. In order to identify general rules, we have mapped replication origins along 1% of the human genome in HeLa cells. We found large gene-poor regions lacking origin and G+C rich regions containing clusters of closely spaced origins. Half of the 283 origins mapped are within or near CpG islands. The connection with gene expression is further reinforced by the observation that most origins overlap with DNAseI hypersensitive sites found at transcriptional regulatory elements. We show, however, that this association is independent of chromatin structure and transcriptional activity. Replication timing analyses coupled to our origin mapping demonstrate that origin dense regions and isolated origins are replicated at every moment in S phase. All together, our data suggest that a relatively strict origin-timing programme regulates DNA replication of the human genome. Keywords: Nascent strands, ENCODE project, HeLAS3 cells, SNS-Chip Four independent preparations of Short Nascent Strands (SNS) were performed. In order to have enough material for microarray hybridisation, we coupled the stringent preparation of SNS with the TLAD method, a technique of linear amplification that can generate several µg of amplified material from 10-20 ng of DNA (Liu et al., 2003).Two were amplified by TLAD (experiments A and B) and hybridized on DNA microarrays, and the other two (experiments C and D) were used for the validation by real-time quantitative PCR (qPCR) of results obtained on micro-arrays. We performed also a gDNA/gDNA hybridization where gDNA are also amplified by TLAD to order to do a control.
2010-05-18 | E-GEOD-10217 | biostudies-arrayexpress