ORC precisely positions nucleosomes at origins of replication
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Matthew Eaton
PROVIDER: E-GEOD-16926 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Conserved nucleosome positioning defines replication origins.
Eaton Matthew L ML Galani Kyriaki K Kang Sukhyun S Bell Stephen P SP MacAlpine David M DM
Genes & development 20100329 8
The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. ...[more]
Similar Datasets
Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.
2014-09-26 | E-GEOD-61743 | 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:The Origin Recognition Complex (ORC) is essential for the initiation of eukaryotic chromosome replication as it loads the replicative helicase, the minichromosome maintenance (MCM) complex, at replication origins. Origins display a stereotypic chromatin structure with nucleosome depletion at ORC binding sites and flanking arrays of regularly spaced nucleosomes. Although discovered long ago, it is unknown how this chromatin struture is established and if it matters for replication. By genome-scale biochemical reconstitution we screened ~300 origins and 17 purified budding yeast chromatin factors and found that ORC established nucleosome depletion over origins and flanking nucleosome arrays by orchestrating the chromatin remodelers INO80, ISW1a, ISW2 and Chd1. The functional importance of ORC’s chromatin organizing activity was directly demonstrated by ORC mutations that maintained classical MCM loader activity but lost array generation activity. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, besides its canonical role as MCM loader, has a second essential function as master regulator of the origin chromatin structure that is a crucial prerequisite for efficient chromosome replication.
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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
Project description:The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell, such as in the orc2-1 mutant, only sites that bind ORC tightly, such as HMR-E, remain fully occupied by ORC, while lower affinity sites, including most origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites) that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication, and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection between ORC, replication origins and cellular metabolism.
2009-11-07 | GSE18895 | 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:Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress. In this study we sought to analyze the chromatin structural changes that take place at sites of DNA replication. To this end, we obtained yeast cell populations synchronously undergoing DNA replication by M-NM-1-factor G1 arrest and release. In order to analyze chromatin structure at sites of DNA replication we first mapped the genomic locations undergoing DNA replication to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Two complementary approaches were taken: (1) we mapped fork positions by chromatin immunoprecipitation (ChIP) of a FLAG-tagged DNA polymerase 1 (Pol1), a replication fork component and (2) we mapped the sites of active DNA synthesis by generating DNA copy number profiles. We then analyzed chromatin structure at the sites of DNA replication by Micrococcal nuclease mononucleosome mapping and by generating histone H3 density maps. We further generated a Normalized Chromatin Accessibility to MNase (NCAM) signal by normalizing MNase mononucleosome signal for histone H3 density. NCAM signal represents a measure of nucleosome accessibility to MNase that is normalized for nucleosome content. These three approaches allowed us to assess potential changes in nucleosome positioning, nucleosome density, and nucleosome accessibility during DNA replication. We searched for changes in chromatin structure by comparing, during S phase, regions undergoing DNA replication to those not yet replicated, and also by comparing the same region before replication (not replicated, G1 arrested control) and during DNA replication in S phase. We typically harvested S phase cells at 30 or 60 minutes after release from G1 arrest. Experimental conditions included releasing cells into rich media at 24M-BM-0C or into rich media containing 200 mM Hydroxyurea (HU). Both conditions slowed down replication fork rate and made these experiments feasible. For each strain, samples for the different experiments (Chromatin for Pol1 and H3 ChIP, in vivo MNase digestion, and DNA for DNA copy number profiles) were harvested simultaneously for each time point. Therefore, comparisons between time points for each strain must be made using samples from the same replicate experiment. Our analysis included WT cells, as well as S phase checkpoint mutants (M-NM-^Tmec1 M-NM-^Tsml1 and mec1-100 M-NM-^Tsml1), as well as control strains (M-NM-^Tsml1).
2013-01-17 | E-GEOD-43548 | biostudies-arrayexpress
Project description:The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition.
2024-03-18 | GSE249817 | GEO
Project description:Our data have revealed a correlation between the timing of ORC and MCM binding to origins and the timing of replication. We hypothesized that origins bind ORC during M with varying affinities, and that delays in ORC binding and pre-RC formation at late-firing origins result in low efficiencies due to these origins subsequently competing less effectively for limiting replication factors. We investigated if equalizing ORC binding results in changes in origin efficiencies. Because the increase in ORC binding occurs during M, we surmised that extending M might result in origins accumulating ORC more equally, leading to more equal distribution of pre-RC and pre-IC assembly among origins. As a consequence, early origins might become less efficient and late origins more efficient. To extend M in cells, we used the drug MBC (Carbendazim), which prevents microtubule polymerization and disrupts the mitotic spindle. Cells were then allowed to undergo S phase in the presence of BrdU, and labeled fragments were isolated by immunoprecipitation of genomic DNA and hybridized to Affymetrix tiling arrays. These results were compared to the replication profile of synchronized cells treated with HU but not with MBC, providing a control for normal origin efficiencies across the genome. This method allows the relative efficiency of origin usage genome-wide to be determined based on signal intensities from array hybridizations. Keywords: comparison between cells treated with a drug and normal cells
2009-03-06 | GSE14864 | GEO