Establishing the program of origin firing during S phase in fission yeast
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE14864 | GEO | 2009/03/06
SECONDARY ACCESSION(S): PRJNA112031
REPOSITORIES: GEO
Similar Datasets
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-2’-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
2007-03-05 | GSE6523 | 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:The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. In this study, we investigate the function of the S. cerevisiae Orc6 subunit, the only subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits pre-replicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 and to load the Mcm2-7 helicase onto origin DNA. We demonstrate that two regions of Orc6 bind Cdt1 directly and that the extreme C-terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of pre-RC assembly. Keywords: ChIP-chip
2007-11-14 | GSE9318 | 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:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157470 | GEO
Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157469 | GEO
Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157468 | 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.
2013-09-30 | GSE48440 | GEO
Project description:In eukaryotes, the hetero-hexameric origin recognition complex (ORC) is responsible for assembling Mcm2-7 complex into a head-to-head double hexamer (DH), forming pre-replicative complex (pre-RC) that licenses origin DNA for replication initiation. This process is tightly controlled throughout the cell cycle to ensure accurate duplication of the genome. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit plays a critical role in promoting pre-RC assembly. We found that removing a short segment (residues 175-200) from Orc2-IDR or mutating a key isoleucine (194) in this region significantly inhibits replication initiation across the genome and causes cell death. Although the Orc2-IDR mutants can still assemble the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate on DNA, the assembled mutant OCCM exhibits impaired ATP hydrolysis, preventing its conversion into Mcm2-7-ORC (MO) complex and subsequent DH formation. Interestingly, our in vitro assays showed that adding the Orc2-IDR peptide in the pre-RC reactions can rescue this defect. Furthermore, phosphorylation of this Orc2-IDR motif by S-cyclin dependent kinase (S-CDK) blocks its binding to Mcm2, leading to defective pre-RC assembly. Our findings provide crucial mechanistic insights into the multifaceted roles of ORC in modulating MCM loading to support origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.
2024-09-18 | GSE262693 | 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