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- PDB-7pmk: S. cerevisiae replisome-SCF(Dia2) complex bound to double-strande... -
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Basic information
Entry | Database: PDB / ID: 7pmk | ||||||||||||
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Title | S. cerevisiae replisome-SCF(Dia2) complex bound to double-stranded DNA (conformation I) | ||||||||||||
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![]() | REPLICATION / Genome stability / DNA replication / Ubiquitination / termination / replisome / cryo-EM / CMG / SCF(Dia2) | ||||||||||||
Function / homology | ![]() mitotic DNA replication termination / maintenance of DNA repeat elements / DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / invasive growth in response to glucose limitation / replication fork arrest / DNA replication initiation / protein-containing complex disassembly / meiotic chromosome segregation ...mitotic DNA replication termination / maintenance of DNA repeat elements / DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / invasive growth in response to glucose limitation / replication fork arrest / DNA replication initiation / protein-containing complex disassembly / meiotic chromosome segregation / 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 / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / 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 / mitotic DNA replication checkpoint signaling / replication fork protection complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / cell cycle / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / SCF ubiquitin ligase complex / nucleotide-excision repair, DNA gap filling / mitotic sister chromatid cohesion / DNA replication proofreading / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / 3'-5' DNA helicase activity / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / replication fork processing / leading strand elongation / nuclear replication fork / regulation of DNA replication / cullin family protein binding / DNA replication origin binding / Dual incision in TC-NER / DNA replication initiation / subtelomeric heterochromatin formation / error-prone translesion synthesis / DNA helicase activity / base-excision repair, gap-filling / meiotic cell cycle / replication fork / helicase activity / 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 / ubiquitin-dependent protein catabolic process / double-stranded DNA binding / DNA helicase / DNA replication / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity / protein ubiquitination / DNA repair / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() Synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||||||||
![]() | Jenkyn-Bedford, M. / Yeeles, J.T.P. / Deegan, T.D. | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1.7 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 13537MC ![]() 7ploC ![]() 7pmnC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA replication licensing factor ... , 5 types, 5 molecules 23467
#1: Protein | Mass: 98911.539 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PACBIOSEQ_LOCUS187, PACBIOSEQ_LOCUS193, PACBIOSEQ_LOCUS195, PACBIOSEQ_LOCUS196, SCNYR20_0007007400, SCP684_0007007100 Production host: ![]() ![]() |
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#2: Protein | Mass: 111987.562 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: ![]() ![]() |
#3: Protein | Mass: 105138.375 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: ![]() ![]() |
#5: Protein | Mass: 113110.211 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM6, YGL201C / Production host: ![]() ![]() |
#6: Protein | Mass: 95049.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM7, PACBIOSEQ_LOCUS429 / Production host: ![]() ![]() |
-Protein , 7 types, 9 molecules 5EFGHKLXY
#4: Protein | Mass: 86505.734 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PACBIOSEQ_LOCUS4054, PACBIOSEQ_LOCUS4112, PACBIOSEQ_LOCUS4129, PACBIOSEQ_LOCUS4153, PACBIOSEQ_LOCUS4202, SCNYR20_0004029000, SCP684_0004028600 Production host: ![]() ![]() | ||||||||
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#11: Protein | Mass: 75154.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: ![]() ![]() | ||||||||
#12: Protein | Mass: 108610.148 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: SCNYR20_0005036600 / Production host: ![]() ![]() #15: Protein | | Mass: 22357.270 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PACBIOSEQ_LOCUS1258, PACBIOSEQ_LOCUS1259, PACBIOSEQ_LOCUS1261, PACBIOSEQ_LOCUS1263, PACBIOSEQ_LOCUS1270, PACBIOSEQ_LOCUS1277, PACBIOSEQ_LOCUS1282, SCNYR20_0001050400, SCP684_0001049800 Production host: ![]() ![]() #16: Protein | | Mass: 85368.844 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: DIA2, YOR080W, YOR29-31 / Production host: ![]() ![]() #19: Protein | | Mass: 141296.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: TOF1, YNL273W, N0636 / Production host: ![]() ![]() #20: Protein | | Mass: 36588.758 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: CSM3, YMR048W, YM9796.01 / Production host: ![]() ![]() |
-DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD
#7: Protein | Mass: 24230.576 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PACBIOSEQ_LOCUS944, PACBIOSEQ_LOCUS956, PACBIOSEQ_LOCUS958, SCNYR20_0001022500, SCP684_0001022000 Production host: ![]() ![]() |
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#8: Protein | Mass: 25096.807 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PACBIOSEQ_LOCUS3163, PACBIOSEQ_LOCUS3191, PACBIOSEQ_LOCUS3224, PACBIOSEQ_LOCUS3231, PACBIOSEQ_LOCUS3255, SCNYR20_0009012300, SCP684_0009011800 Production host: ![]() ![]() |
#9: Protein | Mass: 22004.160 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PACBIOSEQ_LOCUS5150, PACBIOSEQ_LOCUS5239, PACBIOSEQ_LOCUS5244, PACBIOSEQ_LOCUS5270, PACBIOSEQ_LOCUS5331 Production host: ![]() ![]() |
#10: Protein | Mass: 33983.617 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: SLD5, YDR489W / Production host: ![]() ![]() |
-DNA chain , 2 types, 2 molecules IJ
#13: DNA chain | Mass: 37688.645 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others) |
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#14: DNA chain | Mass: 35259.238 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others) |
-DNA polymerase epsilon ... , 2 types, 2 molecules QR
#17: Protein | Mass: 255890.422 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: POL2, DUN2, YNL262W, N0825 / Production host: ![]() ![]() References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters |
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#18: Protein | Mass: 78425.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: DPB2, YPR175W, P9705.7 / Production host: ![]() ![]() |
-Non-polymers , 3 types, 13 molecules ![](data/chem/img/ANP.gif)
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![](data/chem/img/ZN.gif)
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#21: Chemical | #22: Chemical | #23: Chemical | ChemComp-ZN / |
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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 |
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Sample preparation
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.6 | ||||||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||
Specimen support | Details: 15 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Details: Manual plunger |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 400 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / 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: 12730 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2160000 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 369254 Details: Composite map produced from individual cryo-EM density maps (resolutions 3.2 - 4.0 A) using Phenix combine_focused_maps. See publication for details. Symmetry type: POINT |