Establishment and Function of Chromatin Organization at Replication Origins
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE209681 | GEO | 2023/03/30
REPOSITORIES: GEO
Similar Datasets
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:Sumoylation is emerging as a post-translation modification important for chromosome duplication and stability. The origin recognition complex (ORC), which directs DNA replication initiation by loading the MCM replicative helicases onto origins, is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are largely unclear. Here we report the effects of hyper- and hypo-sumoylation of yeast ORC using multiple approaches. We show that ORC hyper-sumoylation preferentially reduces the activity of a subset of early origins, while Orc2 hypo-sumoylation has an opposing effect. Mechanistically, ORC hyper-sumoylation leads to reduced MCM loading in vitro and diminished MCM chromatin association in vivo. The importance of an appropriate level of ORC sumoylation is suggested by the data that either hyper- or hypo-sumoylation of ORC results in genome instability and a dependence on other genome maintenance factors for cell fitness. Thus, yeast ORC sumoylation status needs to be fine-tuned to achieve optimal origin activity control and genome stability.
2023-03-11 | PXD032977 | Pride
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 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:There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, "a site to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.
2023-09-05 | GSE242131 | 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
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:During routine genome duplication, many potential replication origins remain inactive, or dormant. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that can be distinguished from baseline (i.e. ordinarily active) origins by their preferential association with MCM2, a component of the replicative helicase, phosphorylated on serine 108 (pS108-MCM2). pS108-MCM2 is a substrate of the ATR kinase, which is recruited to chromatin via an interaction with hyperacetylated TOPBP1 in cells undergoing replication stress or in cells devoid of SIRT1 deacetylase activity. In turn, S108-MCM2 phosphorylation enhances a second, DDK-dependent, S139-MCM2 phosphorylation, which triggers initiation of DNA replication at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational modifications on the MCM helicase that reflect a balance between SIRT1 activity and ATR signaling.
| MSV000088134 | MassIVE
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