+Open data
-Basic information
Entry | Database: PDB / ID: 7z13 | |||||||||||||||||||||||||||||||||
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Title | S. cerevisiae CMGE dimer nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
Components |
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Keywords | REPLICATION / DNA replication / helicase / initiation / DNA origin | |||||||||||||||||||||||||||||||||
Function / homology | Function and homology information DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex ...DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex / DNA strand elongation involved in mitotic DNA replication / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / SUMO binding / Activation of the pre-replicative complex / CMG complex / single-stranded 3'-5' DNA helicase activity / nuclear pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / Termination of translesion DNA synthesis / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / double-strand break repair via break-induced replication / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / single-stranded DNA helicase activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / mitotic sister chromatid cohesion / DNA replication proofreading / DNA strand elongation involved in DNA replication / leading strand elongation / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / Dual incision in TC-NER / subtelomeric heterochromatin formation / DNA replication initiation / error-prone translesion synthesis / DNA helicase activity / base-excision repair, gap-filling / helicase activity / replication fork / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA helicase / cell cycle / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||||||||||||||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) DNA molecule (others) | |||||||||||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||||||||||||||||||||||||||
Authors | Lewis, J.S. / Sousa, J.S. / Costa, A. | |||||||||||||||||||||||||||||||||
Funding support | European Union, France, United Kingdom, 10items
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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. | |||||||||||||||||||||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7z13.cif.gz | 2.4 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7z13.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7z13.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7z13_validation.pdf.gz | 1.7 MB | Display | wwPDB validaton report |
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Full document | 7z13_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 7z13_validation.xml.gz | 297.7 KB | Display | |
Data in CIF | 7z13_validation.cif.gz | 467.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/z1/7z13 ftp://data.pdbj.org/pub/pdb/validation_reports/z1/7z13 | HTTPS FTP |
-Related structure data
Related structure data | 14439MC 7qhsC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA replication licensing factor ... , 5 types, 10 molecules 2a3b4c6e7f
#1: Protein | Mass: 98911.539 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PACBIOSEQ_LOCUS187, PACBIOSEQ_LOCUS193, PACBIOSEQ_LOCUS195, PACBIOSEQ_LOCUS196, SCNYR20_0007007400, SCP684_0007007100 Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q1S9, DNA helicase #2: Protein | Mass: 111720.242 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase #3: Protein | Mass: 105138.375 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase #5: Protein | Mass: 113110.211 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase #6: Protein | Mass: 95049.875 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM7, PACBIOSEQ_LOCUS429 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q4N0, DNA helicase |
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-Protein , 2 types, 4 molecules 5dEL
#4: Protein | Mass: 86505.734 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PACBIOSEQ_LOCUS4054, PACBIOSEQ_LOCUS4112, PACBIOSEQ_LOCUS4129, PACBIOSEQ_LOCUS4153, PACBIOSEQ_LOCUS4202, SCNYR20_0004029000, SCP684_0004028600 Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PUY8, DNA helicase #11: Protein | Mass: 75154.703 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032 |
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-DNA chain , 2 types, 2 molecules AB
#7: DNA chain | Mass: 16320.634 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others) |
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#8: DNA chain | Mass: 16311.620 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others) |
-DNA replication complex GINS protein ... , 4 types, 8 molecules CJDKHOIP
#9: Protein | Mass: 25718.070 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: PSF3, YOL146W / Production host: Escherichia coli (E. coli) / References: UniProt: Q12146 #10: Protein | Mass: 33983.617 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: SLD5, YDR489W / Production host: Escherichia coli (E. coli) / References: UniProt: Q03406 #13: Protein | Mass: 24230.576 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PACBIOSEQ_LOCUS944, PACBIOSEQ_LOCUS956, PACBIOSEQ_LOCUS958, SCNYR20_0001022500, SCP684_0001022000 Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5Q203 #14: Protein | Mass: 25096.807 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PACBIOSEQ_LOCUS3163, PACBIOSEQ_LOCUS3191, PACBIOSEQ_LOCUS3224, PACBIOSEQ_LOCUS3231, PACBIOSEQ_LOCUS3255, SCNYR20_0009012300, SCP684_0009011800 Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5PX40 |
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-DNA polymerase epsilon ... , 2 types, 4 molecules FMNQ
#12: Protein | Mass: 78425.852 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: DPB2, YPR175W, P9705.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24482 #15: Protein | Mass: 255992.484 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288c / Gene: POL2, DUN2, YNL262W, N0825 / Production host: Saccharomyces cerevisiae (brewer's yeast) References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters |
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-Non-polymers , 4 types, 32 molecules
#16: Chemical | ChemComp-ATP / #17: Chemical | ChemComp-ZN / #18: Chemical | ChemComp-MG / #19: Chemical | ChemComp-ADP / |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: S. cerevisiae CMGE dimer nucleating origin DNA melting Type: COMPLEX Details: Dimeric model reconstituted from symmetry expanded monomer (PDB entry 7QHS). Entity ID: #1-#15 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES 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. |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 4400 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 10 sec. / Electron dose: 1.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 65286 |
Image scans | Movie frames/image: 32 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71348 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT Details: One additional base pair has been built to connect the DNA molecules from the two individual symmetry expanded monomers. | ||||||||||||||||||||||||
Atomic model building |
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