MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1005236 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14350322.fastq.gz | Fastqsanger.gz | |||
| SRR14350323.fastq.gz | Fastqsanger.gz | |||
| SRR14350324.fastq.gz | Fastqsanger.gz | |||
| SRR14350325.fastq.gz | Fastqsanger.gz | |||
| SRR14350326.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 44 |
Similar Datasets
Project description:Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.
| S-EPMC11344781 | biostudies-literature
Project description:The loading and activation of the replicative helicase MCM2-7 are key events during the G1-S phase transition.In budding yeast, the origin recognition complex (ORC) binds to the conserved DNA elements A and B1 of the autonomously replicating sequence (ARS).This is followed by the consecutive loading of two MCM2-7 hetero-hexamers into a MCM2-7 double-hexamer(DH).In S-phase the MCM2-7DH is activated, resulting in two Cdc45-MCM-GINS(CMG) helicases that bidirectionally unwind DNA ahead of the replication fork.Here,we show that MCM2-7 helicase loading across the B1 element displaces ORC fromo rigins.This allows ORC binding and helicase loading at lower affinity binding sites and origins throughout the genome.Furthermore, we mapped the sites of initial DNA unwinding genome-wide and show that these sites appear near the N-terminal domains of the MCM2-7 double-hexamer in proximity of the B1 element.Finally, employing a chemical-biology approach, we establish that during helicase activation the Mcm2/5 interface acts as the DNA exit gate for single-stranded-DNA extrusion.Our work identifies that helicase loading follows a distributive mechanism, allowing for equal MCM2-7 loading across the genome and surprisingly finds that DNA unwinding initiates from the helicase N-terminal interface in proximity to the ARS B1 element.
2024-06-21 | GSE240472 | GEO
Project description:The loading and activation of the replicative helicase MCM2-7 are key events during the G1-S phase transition. In budding yeast, the origin recognition complex (ORC) binds to the conserved DNA elements A and B1 of the autonomously replicating sequence (ARS). This is followed by the consecutive loading of two MCM2-7 hetero-hexamers into a MCM2-7 double-hexamer (DH). In S-phase the MCM2-7 DH is activated, resulting in two Cdc45-MCM-GINS (CMG) helicases that bidirectionally unwind DNA ahead of the replication fork. Here, we show that MCM2-7 helicase loading across the B1 element displaces ORC from origins. This allows ORC binding and helicase loading at lower affinity binding sites and origins throughout the genome. Furthermore, we mapped the sites of initial DNA unwinding genome-wide and show that these sites appear near the N-terminal domains of the MCM2-7 double-hexamer in proximity of the B1 element. Finally, employing a chemical-biology approach, we establish that during helicase activation the Mcm2/5 interface acts as the DNA exit gate for single-stranded-DNA extrusion. Our work identifies that helicase loading follows a distributive mechanism, allowing for equal MCM2-7 loading across the genome and surprisingly finds that DNA unwinding initiates from the helicase N-terminal interface in proximity to the ARS B1 element.
2024-06-21 | GSE173527 | GEO
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 genomes are replicated from many origin sites that are licensed by the loading of inactive double hexamers of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic origins are not irrevocably defined by selection of the loading site for the replicative helicase, but can shift in position after helicase loading. Using purified proteins, we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. In yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a shift in origin positions that correlates with the direction of transcription. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict. 4 samples: one replicate for WT at 37C, two replicates for rat1-1 at 37C, and one replicate for rat1-1 at 24C. All are single-end sequenced via Ion Torrent PGM methodology. rat1 = Nuclear 5' to 3' single-stranded RNA exonuclease; involved in RNA metabolism (http://www.yeastgenome.org/locus/S000005574/overview). 4 ChIP seq samples and their duplicates are submitted. rat1-1 ORC ChIP at 24C and 37C; rat1-1 MCM ChIP at 24C and 37C.
2016-01-07 | E-GEOD-69065 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2024-06-18 | GSE240779 | 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 origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with several subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decrease ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.
2022-10-19 | GSE199896 | GEO
Project description:Nucleoporins facilitate ORC loading onto chromatin
| PRJNA821897 | ENA
Project description:The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decreased ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.
2022-10-19 | PXD033045 | Pride