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Open data
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Basic information
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Title | S. cerevisiae CMGE dimer nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
![]() | Consensus map of CMGE dimer model generated from EMD EMD-13978 | |||||||||||||||||||||||||||||||||
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Function / homology | ![]() DNA-templated DNA replication maintenance of fidelity / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||||||||||||||||||||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||||||||||||||||||||||||||
Method | ![]() ![]() | |||||||||||||||||||||||||||||||||
![]() | Lewis JS / Sousa JS / Costa A | |||||||||||||||||||||||||||||||||
Funding support | European Union, ![]() ![]()
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![]() | ![]() Title: Mechanism of replication origin melting nucleated by CMG helicase assembly. Authors: Jacob S Lewis / Marta H Gross / Joana Sousa / Sarah S Henrikus / Julia F Greiwe / Andrea Nans / John F X Diffley / Alessandro Costa / ![]() Abstract: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex ...The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation. | |||||||||||||||||||||||||||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 43.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 39.3 KB 39.3 KB | Display Display | ![]() |
Images | ![]() | 83.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7z13MC ![]() 7qhsC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||
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Annotation | Consensus map of CMGE dimer model generated from EMD EMD-13978 | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
+Entire : S. cerevisiae CMGE dimer nucleating origin DNA melting
+Supramolecule #1: S. cerevisiae CMGE dimer nucleating origin DNA melting
+Macromolecule #1: DNA replication licensing factor MCM2
+Macromolecule #2: DNA replication licensing factor MCM3
+Macromolecule #3: DNA replication licensing factor MCM4
+Macromolecule #4: DNA helicase
+Macromolecule #5: DNA replication licensing factor MCM6
+Macromolecule #6: DNA replication licensing factor MCM7
+Macromolecule #9: DNA replication complex GINS protein PSF3
+Macromolecule #10: DNA replication complex GINS protein SLD5
+Macromolecule #11: Cell division control protein 45
+Macromolecule #12: DNA polymerase epsilon subunit B
+Macromolecule #13: DNA replication complex GINS protein PSF1
+Macromolecule #14: DNA replication complex GINS protein PSF2
+Macromolecule #15: DNA polymerase epsilon catalytic subunit A
+Macromolecule #7: DNA (53-MER)
+Macromolecule #8: DNA (53-MER)
+Macromolecule #16: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #17: ZINC ION
+Macromolecule #18: MAGNESIUM ION
+Macromolecule #19: ADENOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
Details | four microlitres of sample was applied on a grid and incubated for 2 min at room temperature before blotting with filter paper for 5.5 s and plunge-freezing in liquid ethane. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 65286 / Average exposure time: 10.0 sec. / Average electron dose: 1.6 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
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Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 71348 |