H2020 Marie Curie Actions of the European Commission
European Union
European Molecular Biology Organization (EMBO)
1177-2020
European Union
Human Frontier Science Program (HFSP)
LT000834/2020-L
European Union
European Molecular Biology Organization (EMBO)
962-2019
European Union
The Francis Crick Institute
FC001065
European Union
The Francis Crick Institute
FC001066
European Union
Wellcome Trust
106252/Z/14/Z
European Union
European Research Council (ERC)
669424-CHROMOREP
European Union
European Research Council (ERC)
820102
European Union
Citation
Journal: Nature / Year: 2022 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.
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
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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
Particle selection
Number selected: 927109
Startup model
Type of model: INSILICO MODEL
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 49766
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