+Open data
-Basic information
Entry | Database: PDB / ID: 7plo | ||||||||||||
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Title | H. sapiens replisome-CUL2/LRR1 complex | ||||||||||||
Components |
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Keywords | REPLICATION / Genome stability / DNA replication / Ubiquitination / termination / replisome / cryo-EM / CMG / Cul2LRR1 / DNA / polymerase / helicase | ||||||||||||
Function / homology | Function and homology information cellular response to bleomycin / DNA secondary structure binding / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Switching of origins to a post-replicative state / Unwinding of DNA / cell cycle phase transition / cellular response to cisplatin / DNA replication initiation ...cellular response to bleomycin / DNA secondary structure binding / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Switching of origins to a post-replicative state / Unwinding of DNA / cell cycle phase transition / cellular response to cisplatin / DNA replication initiation / epsilon DNA polymerase complex / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / cellular response to hydroxyurea / anaphase-promoting complex binding / cullin-RING-type E3 NEDD8 transferase / alpha DNA polymerase:primase complex / mitotic DNA replication / cellular response to chemical stress / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / CMG complex / DNA replication checkpoint signaling / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / nucleotide-excision repair, DNA gap filling / single-stranded DNA 3'-5' DNA exonuclease activity / DNA replication preinitiation complex / target-directed miRNA degradation / MCM complex / regulation of phosphorylation / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / replication fork protection complex / elongin complex / DNA replication proofreading / VCB complex / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / positive regulation of protein autoubiquitination / protein neddylation / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / NEDD8 ligase activity / Cul5-RING ubiquitin ligase complex / positive regulation of double-strand break repair / negative regulation of response to oxidative stress / DNA strand elongation involved in DNA replication / ubiquitin-ubiquitin ligase activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / inner cell mass cell proliferation / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / negative regulation of type I interferon production / ubiquitin ligase complex scaffold activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul4B-RING E3 ubiquitin ligase complex / Cul3-RING ubiquitin ligase complex / activation of protein kinase activity / branching morphogenesis of an epithelial tube / DNA synthesis involved in DNA repair / cochlea development / G1/S-Specific Transcription / leading strand elongation / DNA unwinding involved in DNA replication / Apoptotic cleavage of cellular proteins / Prolactin receptor signaling / replication fork processing / nuclear replication fork / protein monoubiquitination / mitotic G2 DNA damage checkpoint signaling / DNA replication origin binding / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / cullin family protein binding / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / positive regulation of double-strand break repair via homologous recombination / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / DNA replication initiation / Tat-mediated elongation of the HIV-1 transcript / error-prone translesion synthesis / embryonic organ development / Formation of HIV-1 elongation complex containing HIV-1 Tat / cellular response to interleukin-4 / Formation of HIV elongation complex in the absence of HIV Tat / protein K48-linked ubiquitination / Activation of ATR in response to replication stress / RNA Polymerase II Transcription Elongation / Nuclear events stimulated by ALK signaling in cancer / Formation of RNA Pol II elongation complex / response to UV / RNA Polymerase II Pre-transcription Events / base-excision repair, gap-filling / DNA helicase activity / positive regulation of TORC1 signaling Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å | ||||||||||||
Authors | Jones, M.J. / Yeeles, J.T.P. / Deegan, T.D. / Jenkyn-Bedford, M. | ||||||||||||
Funding support | United Kingdom, 3items
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Citation | Journal: Nature / Year: 2021 Title: A conserved mechanism for regulating replisome disassembly in eukaryotes. Authors: Michael Jenkyn-Bedford / Morgan L Jones / Yasemin Baris / Karim P M Labib / Giuseppe Cannone / Joseph T P Yeeles / Tom D Deegan / Abstract: Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily ...Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF in budding yeast, CUL2 in metazoa), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA. However, it is unknown how SCF and CUL2 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7plo.cif.gz | 3.4 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7plo.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7plo.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7plo_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 7plo_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 7plo_validation.xml.gz | 222.4 KB | Display | |
Data in CIF | 7plo_validation.cif.gz | 365.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pl/7plo ftp://data.pdbj.org/pub/pdb/validation_reports/pl/7plo | HTTPS FTP |
-Related structure data
Related structure data | 13494MC 7pmkC 7pmnC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA replication licensing factor ... , 6 types, 6 molecules 234567
#1: Protein | Mass: 102034.102 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM2, BM28, CCNL1, CDCL1, KIAA0030 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49736, DNA helicase |
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#2: Protein | Mass: 91110.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P25205, DNA helicase |
#3: Protein | Mass: 96684.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM4, CDC21 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33991, DNA helicase |
#4: Protein | Mass: 82406.633 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM5, CDC46 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33992, DNA helicase |
#5: Protein | Mass: 93010.273 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM6 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14566, DNA helicase |
#6: Protein | Mass: 81411.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM7, CDC47, MCM2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33993, DNA helicase |
-DNA polymerase epsilon ... , 2 types, 2 molecules AB
#7: Protein | Mass: 59600.887 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLE2, DPE2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P56282 |
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#8: Protein | Mass: 261855.266 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLE, POLE1 / Production host: Trichoplusia ni (cabbage looper) References: UniProt: Q07864, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters |
-Protein , 10 types, 12 molecules CHIJKLOPQRST
#9: Protein | Mass: 66016.891 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CDC45, CDC45L, CDC45L2, UNQ374/PRO710 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75419 | ||||||||||||||||
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#14: Protein | Mass: 130098.148 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: WDHD1, AND1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75717 #15: Protein | | Mass: 138903.031 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TIMELESS, TIM, TIM1, TIMELESS1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UNS1 #16: Protein | | Mass: 34600.223 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TIPIN / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BVW5 #19: Protein | | Mass: 46789.648 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LRR1, PPIL5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q96L50 #20: Protein | | Mass: 13147.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ELOB, TCEB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15370 #21: Protein | | Mass: 155184.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CLSPN / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9HAW4 #22: Protein | | Mass: 12485.135 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ELOC, TCEB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15369 #23: Protein | | Mass: 87098.930 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CUL2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13617 #24: Protein | | Mass: 12289.977 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Trichoplusia ni (cabbage looper) References: UniProt: P62877, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase |
-DNA replication complex GINS protein ... , 4 types, 4 molecules DEFG
#10: Protein | Mass: 23022.469 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS1, KIAA0186, PSF1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14691 |
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#11: Protein | Mass: 21453.713 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS2, PSF2, CGI-122, DC5, HSPC037 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y248 |
#12: Protein | Mass: 24562.611 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS3, PSF3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BRX5 |
#13: Protein | Mass: 30114.236 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS4, SLD5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BRT9 |
-DNA chain , 2 types, 2 molecules MN
#17: DNA chain | Mass: 26396.836 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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#18: DNA chain | Mass: 12279.929 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
-Non-polymers , 4 types, 15 molecules
#25: Chemical | ChemComp-ZN / #26: Chemical | #27: Chemical | #28: Chemical | ChemComp-SO4 / | |
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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: Human replisome engaged with CUL2-LRR1 / Type: COMPLEX / Entity ID: #21, #1-#20, #22-#24 / Source: MULTIPLE SOURCES |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Trichoplusia ni (cabbage looper) |
Buffer solution | pH: 7.6 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | 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: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm / Cs: 0.1 mm / C2 aperture diameter: 2.7 µm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 4 sec. / Electron dose: 38.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 16721 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2412000 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 232000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT |