Nucleoporins facilitate ORC loading onto chromatin
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ABSTRACT:
PROVIDER: PRJNA821897 | ENA |
REPOSITORIES: ENA
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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: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
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: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:MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing
| PRJNA1005236 | ENA
Project description:A Role for Chromatin Remodeling in Cohesin Loading onto Chromosomes
| PRJNA483480 | ENA
Project description:During DNA replication initiation, the Origin Recognition Complex (ORC) and Cdc6 co-associate on DNA to load replicative helicases onto origins of replication. We apply DSSO crosslinking mass spectrometry of reconstituted ORC-DNA-Cdc6 to identify interactions between different subunits and domains in the complex.
2022-02-28 | PXD031033 | Pride
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:DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.
| S-EPMC7395460 | biostudies-literature
Project description:Map ORC binding sites to identify replication origins in C. albicans by using polyclonal ORC antibodies (gift from Stephen Bell Lab). Due to the unsynchronized nature of Candida cells, log-phase cultures were taken to perfoem ChIP-chip experiments to find the genome-wide ORC binding sites.
2014-02-13 | GSE54923 | GEO