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- PDB-6s2e: Cryo-EM structure of Ctf18-1-8 in complex with the catalytic doma... -
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Basic information
Entry | Database: PDB / ID: 6s2e | |||||||||
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Title | Cryo-EM structure of Ctf18-1-8 in complex with the catalytic domain of DNA polymerase epsilon | |||||||||
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![]() | REPLICATION / DNA polymerase / PCNA loader / protein complex | |||||||||
Function / homology | ![]() maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / Ctf18 RFC-like complex / telomere tethering at nuclear periphery / maintenance of DNA trinucleotide repeats / SUMO binding / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity ...maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / Ctf18 RFC-like complex / telomere tethering at nuclear periphery / maintenance of DNA trinucleotide repeats / SUMO binding / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / mitotic sister chromatid cohesion / DNA replication proofreading / leading strand elongation / nuclear replication fork / chromosome, centromeric region / Dual incision in TC-NER / DNA replication initiation / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / double-strand break repair via homologous recombination / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / double-stranded DNA binding / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / mRNA binding / nucleotide binding / chromatin / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / ATP binding / nucleus Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
![]() | Grabarczyk, D.B. / Song, B. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication. Authors: Katy Stokes / Alicja Winczura / Boyuan Song / Giacomo De Piccoli / Daniel B Grabarczyk / ![]() ![]() Abstract: The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA ...The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability. | |||||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 301.1 KB | Display | ![]() |
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PDB format | ![]() | 228.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 10088MC ![]() 6s1cC ![]() 6s2fC 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
#1: Protein | Mass: 139618.359 Da / Num. of mol.: 1 / Mutation: D290A, E292A Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters |
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#2: Protein | Mass: 15189.688 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#3: Protein/peptide | Mass: 3859.329 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#4: Protein | Mass: 44133.785 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#5: Chemical | ChemComp-SF4 / |
Has ligand of interest | N |
-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
Component | Name: Complex of the catalytic domain of DNA polymerase epsilon with the Ctf18-1-8 module of Ctf18-RFC Type: COMPLEX / Entity ID: #2-#4 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.2 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.1 mg/ml / 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 R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % |
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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 defocus max: 2600 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 75 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) |
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Processing
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93191 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient | ||||||||||||||||||||||||||||||||
Atomic model building |
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Refinement | Highest resolution: 4.2 Å / Stereochemistry target values: CDL v1.2 | ||||||||||||||||||||||||||||||||
Refine LS restraints |
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