Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA102981 | ENA |
REPOSITORIES: ENA
Similar Datasets
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: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:Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1
| PRJNA155861 | ENA
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 minichromosome maintenance complex (MCM) DNA helicase is an important replicative factor during DNA replication. The proper chromatin loading of MCM is a key step to ensure replication initiation during G1/S phase. Because replication initiation is regulated by multiple biological cues, additional changes to MCM may provide deeper understanding towards this event. Here, we uncover that the histidine methyltransferase SETD3 promotes DNA replication in an enzymatic activity dependent manner. Nascent-strand sequencing (NS-seq) shows that SETD3 regulates replication initiation, as depletion of SETD3 attenuates early replication origins firing. Mechanistically, biochemical experiments reveal that SETD3 binds MCM mainly during G1/S phase, which is required for CDT1-mediated chromatin loading of MCM. The MCM loading relies on the histidine-459 methylation (H459me) on MCM7 that is catalyzed by SETD3. Impairment of H459 methylation attenuates DNA synthesis and chromatin loading of MCM. Furthermore, we show that CDK2 phosphorylates SETD3 at Serine-21 during the G1/S phase, which is required for DNA replication and cell cycle progression. These findings demonstrate a novel mechanism by which SETD3 methylates MCM to regulate replication initiation.
2024-03-12 | GSE243937 | GEO
Project description:Dynamic mRNA gene expression during a nutritional downshift from glutamine to proline
| PRJNA237815 | ENA
Project description:Dynamic mRNA gene expression during a nutritional upshift from proline to glutamine
| PRJNA237816 | ENA
Project description:Differential Recruitment of the Splicing Machinery during Transcription Predicts Genome-wide Patterns of mRNA Splicing
| PRJNA98723 | ENA
Project description:Dynamic Sumoylation of a Conserved Transcription Corepressor Prevents Persistent Inclusion Formation during Hyperosmotic Stress
| PRJNA246545 | ENA