Evaluation of ORC and Mcm2-7 Distribution Throughout the Drosophila Kc Cell Cycle
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ABSTRACT:
PROVIDER: PRJNA269387 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:This SuperSeries is composed of the SubSeries listed below and previous submissions made by modENCODE (GSE17282, GSE17283, and GSE41349).
2014-12-05 | GSE63915 | 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 MCM2-7DH 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:MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing
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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:Determination of the genome-wide distribution of the Mcm2-7 helicase by chromatin immunoprecipitation in the Drosophila Kc167 cell line at the beginning of S phase. Kc167 cells were arrested at the G1/S transition with hydroxyurea (HU). Goal was to evaluate changes in the genome-wide Mcm2-7 distribution throughout the cell cycle, and how this relates to the excess Mcm2-7 loaded onto chromatin in G1. ChIP-Chip of Mcm2-7 in HU compared to input genomic DNA. Biological Replicates: 2
2014-11-27 | E-GEOD-63680 | biostudies-arrayexpress
Project description:Determination of the genome-wide distribution of the Mcm2-7 helicase by chromatin immunoprecipitation in the Drosophila Kc167 cell line at the beginning of S phase. Kc167 cells were arrested at the G1/S transition with hydroxyurea (HU). Goal was to evaluate changes in the genome-wide Mcm2-7 distribution throughout the cell cycle, and how this relates to the excess Mcm2-7 loaded onto chromatin in G1.
2014-11-27 | GSE63680 | 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.
2010-03-19 | GSE16926 | 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:Determination of the genome-wide distribution of the Mcm2-7 helicase by chromatin immunoprecipitation in the Drosophila Kc167 cell line at the end of S phase. Kc167 cells were synchronized by first arresting at the G1/S transition with hydroxyurea (HU), and then releasing from the HU arrest point for 6 hours. Goal was to evaluate changes in the genome-wide Mcm2-7 distribution throughout the cell cycle, and how this relates to the excess Mcm2-7 loaded onto chromatin in G1.
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