+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 / Switching of origins to a post-replicative state / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / cell cycle phase transition ...cellular response to bleomycin / DNA secondary structure binding / Switching of origins to a post-replicative state / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / cell cycle phase transition / cellular response to cisplatin / GINS complex / DNA strand elongation involved in mitotic DNA replication / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / cellular response to hydroxyurea / alpha DNA polymerase:primase complex / mitotic DNA replication / cullin-RING-type E3 NEDD8 transferase / anaphase-promoting complex binding / cellular response to chemical stress / NEDD8 transferase activity / CMG complex / cullin-RING ubiquitin ligase complex / DNA replication checkpoint signaling / single-stranded 3'-5' DNA helicase activity / entrainment of circadian clock / single-stranded DNA 3'-5' DNA exonuclease activity / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / regulation of phosphorylation / MCM complex / DNA replication preinitiation complex / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / target-directed miRNA degradation / elongin complex / VCB complex / positive regulation of protein autoubiquitination / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / protein neddylation / double-strand break repair via break-induced replication / NEDD8 ligase activity / mitotic intra-S DNA damage checkpoint signaling / positive regulation of double-strand break repair / Cul5-RING ubiquitin ligase complex / negative regulation of response to oxidative stress / regulation of DNA-templated DNA replication initiation / ubiquitin-ubiquitin ligase activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / Cul4A-RING E3 ubiquitin ligase complex / single-stranded DNA helicase activity / SCF ubiquitin ligase complex / inner cell mass cell proliferation / Cul2-RING ubiquitin ligase complex / negative regulation of type I interferon production / nucleotide-excision repair, DNA gap filling / Cul4B-RING E3 ubiquitin ligase complex / DNA replication proofreading / ubiquitin ligase complex scaffold activity / DNA strand elongation involved in DNA replication / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / branching morphogenesis of an epithelial tube / DNA synthesis involved in DNA repair / cochlea development / G1/S-Specific Transcription / activation of protein kinase activity / leading strand elongation / replication fork processing / Prolactin receptor signaling / Apoptotic cleavage of cellular proteins / DNA unwinding involved in DNA replication / nuclear replication fork / protein monoubiquitination / mitotic G2 DNA damage checkpoint signaling / 3'-5' DNA helicase activity / 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 / Activation of the pre-replicative complex / PCNA-Dependent Long Patch Base Excision Repair / DNA replication initiation / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / embryonic organ development / cellular response to interleukin-4 / protein K48-linked ubiquitination / Formation of HIV elongation complex in the absence of HIV Tat / error-prone translesion synthesis / Nuclear events stimulated by ALK signaling in cancer / Activation of ATR in response to replication stress / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / response to UV 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 |