Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6.

ABSTRACT: 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.

SUBMITTER: Yuan Z

PROVIDER: S-EPMC7395460 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6.

Project description:The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

| S-EPMC8222357 | biostudies-literature

Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1.

Project description:To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

| S-EPMC5503505 | biostudies-literature

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.

Project description:In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-?S, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.

| S-EPMC3735830 | biostudies-literature

ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.

Project description:Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.

| S-EPMC2952294 | biostudies-literature

mTOR Signaling Upregulates CDC6 via Suppressing miR-3178 and Promotes the Loading of DNA Replication Helicase.

Project description:mTOR signaling pathway is deregulated in most cancers and uncontrolled cell cycle progression is a hallmark of cancer cell. However, the precise molecular mechanisms of the regulation of DNA replication and chromatin metabolism by mTOR signaling are largely unknown. We herein report that mTOR signaling promotes the loading of MCM2-7 helicase onto chromatin and upregulates DNA replication licensing factor CDC6. Pharmacological inhibition of mTOR kinase resulted in CHK1 checkpoint activation and decreased MCM2-7 replication helicase and PCNA associated with chromatins. Further pharmacological and genetic studies demonstrated CDC6 is positively controlled by mTORC1-S6K1 and mTORC2 signaling. miRNA screening revealed mTOR signaling suppresses miR-3178 thereby upregulating CDC6. Analysis of TCGA data found that CDC6 is overexpressed in most cancers and associates with the poor survival of cancer patients. Our findings suggest that mTOR signaling may control DNA replication origin licensing and replisome stability thereby cell cycle progression through CDC6 regulation.

| S-EPMC6614418 | biostudies-literature

A helicase-tethered ORC flip enables bidirectional helicase loading.

Project description:Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin-recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.

| S-EPMC8828053 | biostudies-literature

Nucleoporins facilitate ORC loading onto chromatin

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

Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy.

Project description:The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATP?S. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.

| S-EPMC3299985 | biostudies-literature

Nucleoporins facilitate ORC loading onto chromatin

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

The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.

Project description:DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2-7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2-7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.

| S-EPMC6547688 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data