Ordered and disordered regions of the Origin Recognition Complex direct differential in vivo binding at distinct motif sequences.
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE249817 | GEO | 2024/03/18
REPOSITORIES: GEO
Similar Datasets
Project description:The evolutionarily conserved Origin Recognition Complex (ORC), plays a key role in origin selection in eukaryotes. However, ORC is strikingly divergent in its DNA binding specificity, ranging from base-specific interactions in Saccharomyces cerevisiae to base-agnostic interactions in humans. The mechanisms underlying this distinct selectivity is unknown. Atomic model of the yeast ORC showed that base-specific interaction with the invariant thymines of the ARS consensus sequence (ACS) is encoded by a 19-amino acid insertion helix (IH) embedded in the winged helix domain (WHD) of Orc4. This IH is absent in the Orc4 of metazoans including humans, suggesting that removal of the IH might give the yeast ORC “human-like” properties. Indeed, yeast strain engineered with IH deficient Orc4 has completely altered ORC-binding sites enriched in poly-dT tracts located in larger nucleosome-depleted and intergenic open chromatin. In vivo and in vitro assays show that the mutant ORC loads MCM efficiently, in spite of its altered specificity in favor of binding patterns more characteristic of those observed in humans/metazoans. This work provides insights for understanding how ORC evolves to adopt a life cycle that requires plasticity in origin selection during development.
2020-10-18 | GSE149163 | GEO
Project description:The budding yeast genome is marked by 250-350 origins of DNA replication. These origins are bound by the origin recognition complex (ORC) throughout the cell cycle. ORC has known DNA binding sequence preferences which, though necessary for binding, are not sufficient to fully specify a genomic locus as being bound by ORC, indicating that the cell must use additional chromosomal cues to specify ORC binding sites and origins of replication. Using high-throughput sequencing to precisely locate both ORC binding sites and nucleosome locations genome-wide, we find that a nucleosome depleted region (NDR) and precisely positioned nucleosomes are a ubiquitous feature of yeast replication origins. The ARS consensus sequence (ACS) and adjacent sequences are sufficient to maintain the nucleosome-free properties of the NDR. We use a temperature sensitive ORC1 mutant to demonstrate that ORC is required to maintain precisely positioned nucleosomes at origins of replication. These findings demonstrate the importance of local nucleosome positioning at replication origins, and that chromatin organization is an important determinant of origin selection. Examination of nucleosome positioning in wild-type and orc1-161ts mutant S. cerevisiae at room temperature and heatshock temperatures. Examination of ORC binding locations by ChIP-seq. All reported coordinates are based on the SGD genome build released 12/16/2005.
2010-03-19 | E-GEOD-16926 | biostudies-arrayexpress
Project description:The budding yeast genome is marked by 250-350 origins of DNA replication. These origins are bound by the origin recognition complex (ORC) throughout the cell cycle. ORC has known DNA binding sequence preferences which, though necessary for binding, are not sufficient to fully specify a genomic locus as being bound by ORC, indicating that the cell must use additional chromosomal cues to specify ORC binding sites and origins of replication. Using high-throughput sequencing to precisely locate both ORC binding sites and nucleosome locations genome-wide, we find that a nucleosome depleted region (NDR) and precisely positioned nucleosomes are a ubiquitous feature of yeast replication origins. The ARS consensus sequence (ACS) and adjacent sequences are sufficient to maintain the nucleosome-free properties of the NDR. We use a temperature sensitive ORC1 mutant to demonstrate that ORC is required to maintain precisely positioned nucleosomes at origins of replication. These findings demonstrate the importance of local nucleosome positioning at replication origins, and that chromatin organization is an important determinant of origin selection.
2010-03-19 | GSE16926 | GEO
Project description:Chromatin Immunoprecipitation followed by high throughput sequencing to identify the location of Orc1, Orc2 and Orc4 genomic enrichment.
2021-07-02 | GSE165127 | GEO
Project description:DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2â-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Early and late origins tend to distribute separately in large chromosome regions. Inactivation of replication checkpoint by deletion of Cds1 resulted in BrdU incorporation with HU specifically at the late origins. An origin-exchange experiment showed that replication timing was not intrinsic to origin but dependent on its surrounding region. Interestingly, pericentromeric heterochromatin and the silent mating type locus replicated in the presence of HU, while the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where ORC was located. Thus, replication is differentially regulated in chromosome domains. Keywords: ChIP-chip analysis Experiment Design: ⢠The goal of the experiment â Genome-wide localization of ORC and MCM binding sites and identification of replication origins in Shizosaccaromyces pombe ⢠A brief description of the experiment (e.g., the abstract from the related publication) DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2â-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Early and late origins tend to distribute separately in large chromosome regions. Inactivation of replication checkpoint by deletion of Cds1 resulted in BrdU incorporation with HU specifically at the late origins. An origin-exchange experiment showed that replication timing was not intrinsic to origin but dependent on its surrounding region. Interestingly, pericentromeric heterochromatin and the silent mating type locus replicated in the presence of HU, while the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where ORC was located. Thus, replication is differentially regulated in chromosome domains. ⢠Keywords, for example, time course, cell type comparison, array CGH (the use of MGED ontology terms is recommended). Mitosis, Cell cycle, Schizosaccharomyces pombe, Whole-genome, Tiling array, Chromosome II, III array, Orc1, Orc4, Mcm6, BrdU-labeling, cds1Î. ⢠Experimental factors Distribution of the Orc1 and Mcm6 in WT in G1 phase (S. pombe). Distribution of the Orc4 in WT in asynchronous cells (S. pombe). Distribution of BrdU-incorporated DNA in WT and in cds1 deletion mutant in S phase in the presence of HU (S. pombe). ⢠Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Pombe whole-genome tiling array were used. BrdU analysis: Hybridization data for BrdU fraction (replicated DNA) was compared with Whole fraction (unreplicated DNA) or hybridization data for BrdU fraction of cds1Î cells was compared with that of WT cells. Pombe whole-genome tiling array were used. ⢠Quality control steps taken Different subunits of the same complex. Confirmation of several loci by q-PCR. Checking the BrdU-labeled DNA fraction in CsCl gradient by q-PCR or slot-blot analysis. ⢠Links to the publication, any supplemental websites or database accession numbers. GSE6523 Samples used, extract preparation and labelling: ⢠The origin of each biological sample ChIP analysis: Schizosaccharomyces pombe nda3-KM311 cdc10-129 strain harboring pREP81-cdc18 and pREP42-cdt1. BrdU analysis: Schizosaccharomyces pombe wild type (cdc25-22 nmt1-TK) and cds1Î strains. ⢠Manipulation of biological samples and protocols used ChIP analysis: Chromatin immunoprecipitation (ChIP) in G1 arrested cells or asynchronous cells were performed as previously described (Takahashi et al., 2003, EMBO). Hybridization to Affimetrix high-density oligonucleotide array of S. pombe chromosomes 2 and 3 and whole genome tiling array of S. pombe was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). BrdU analysis: Growing cdc25-22 cells expressing Thymidine Kinase were arrested at G2/M boundary and released in the presence of BrdU and HU. BrdU was incorporated in nascent DNA in following S phase. The genomic DNA was digested by HaeIII and BrdU-labeled DNA was purified in CsCl density gradient centrifugation. Hybridization to Affimetrix high-density oligonucleotide array of S. pombe whole genome tiling array was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature).
2010-06-11 | E-GEOD-6523 | biostudies-arrayexpress
Project description:Orc1, Orc2 and Orc4 ChIP-seq in Torulaspora delbrueckii
| PRJNA693306 | ENA
Project description:Pch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific ‘licensing’ of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1 and Rec8). This suggests that although Pch2 is linked to axis/SC-directed recruitment and function, the chromosomal population of Pch2 described here is not directly associated with chromosomal axis sites. In line with this observation, interfering with the pool of Pch2 that associates with active RNAPII transcription does not lead to effects on the chromosomal abundance of Hop1, a known axial client of Pch2. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription.
2020-06-14 | GSE138429 | GEO
Project description:Pch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific ‘licensing’ of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1 and Rec8). This suggests that although Pch2 is linked to axis/SC-directed recruitment and function, the chromosomal population of Pch2 described here is not directly associated with chromosomal axis sites. In line with this observation, interfering with the pool of Pch2 that associates with active RNAPII transcription does not lead to effects on the chromosomal abundance of Hop1, a known axial client of Pch2. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription.
2020-06-14 | GSE144835 | GEO
Project description:Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level. Analysis of ORC genomic DNA binding across three ORC concentrations.
2013-09-30 | E-GEOD-48440 | biostudies-arrayexpress
Project description:Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level.
2013-09-30 | GSE48440 | GEO