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Yorodumi- PDB-6hv8: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant -
+Open data
-Basic information
Entry | Database: PDB / ID: 6hv8 | ||||||||||||
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Title | Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant | ||||||||||||
Components |
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Keywords | DNA BINDING PROTEIN / Polymerase epsilon / DNA replication / enzyme / DNA polymerase | ||||||||||||
Function / homology | Function and homology information DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling ...DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / single-stranded DNA binding / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / cell cycle / nucleotide binding / mRNA binding / DNA binding / zinc ion binding / nucleus / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å | ||||||||||||
Authors | Goswami, P. / Purkiss, A. / Cheung, A. / Costa, A. | ||||||||||||
Funding support | United Kingdom, 3items
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Citation | Journal: Nat Commun / Year: 2018 Title: Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome. Authors: Panchali Goswami / Ferdos Abid Ali / Max E Douglas / Julia Locke / Andrew Purkiss / Agnieszka Janska / Patrik Eickhoff / Anne Early / Andrea Nans / Alan M C Cheung / John F X Diffley / Alessandro Costa / Abstract: Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, ...Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro. | ||||||||||||
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 | 6hv8.cif.gz | 225.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6hv8.ent.gz | 176.5 KB | Display | PDB format |
PDBx/mmJSON format | 6hv8.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/hv/6hv8 ftp://data.pdbj.org/pub/pdb/validation_reports/hv/6hv8 | HTTPS FTP |
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-Related structure data
Related structure data | 0287MC 0288C 6hv9C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 78408.758 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: DPB2, YPR175W, P9705.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24482, DNA-directed DNA polymerase |
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#2: Protein | Mass: 104984.844 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: POL2, DUN2, YNL262W, N0825 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P21951, DNA-directed DNA polymerase |
#3: Chemical |
-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: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2) Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Buffer solution | pH: 7.6 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
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 FIELDBright-field microscopy |
Image recording | Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) |
-Processing
Image processing | Details: Volta phase plate |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
Symmetry | Point symmetry: C1 (asymmetric) |
3D reconstruction | Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161376 / Symmetry type: POINT |