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- PDB-6hv8: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant -

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Entry
Database: PDB / ID: 6hv8
TitleCryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant
Components
  • DNA polymerase epsilon catalytic subunit A
  • DNA polymerase epsilon subunit B
KeywordsDNA BINDING PROTEIN / Polymerase epsilon / DNA replication / enzyme / DNA polymerase
Function / homology
Function and homology information


Telomere C-strand synthesis initiation / Termination of translesion DNA synthesis / Dual Incision in GG-NER / Dual incision in TC-NER / DNA replication initiation / Activation of the pre-replicative complex / DNA-dependent DNA replication maintenance of fidelity / gene conversion / epsilon DNA polymerase complex / DNA replication proofreading ...Telomere C-strand synthesis initiation / Termination of translesion DNA synthesis / Dual Incision in GG-NER / Dual incision in TC-NER / DNA replication initiation / Activation of the pre-replicative complex / DNA-dependent DNA replication maintenance of fidelity / gene conversion / epsilon DNA polymerase complex / DNA replication proofreading / heterochromatin organization involved in chromatin silencing / single-stranded DNA 3'-5' exodeoxyribonuclease activity / mitotic DNA replication checkpoint / intra-S DNA damage checkpoint / leading strand elongation / mitotic sister chromatid cohesion / base-excision repair, gap-filling / replication fork / nucleotide-excision repair, DNA gap filling / double-strand break repair / error-prone translesion synthesis / base-excision repair / DNA-dependent DNA replication / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / mitotic cell cycle / double-strand break repair via nonhomologous end joining / double-stranded DNA binding / ec:2.7.7.7: / cell cycle / mRNA binding / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / zinc ion binding / nucleus / cytoplasm
DNA polymerase family B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase family B, exonuclease domain / Domain of unknown function (DUF1744) / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B / DNA polymerase alpha/delta/epsilon, subunit B / Ribonuclease H-like superfamily / DNA polymerase epsilon, subunit B ...DNA polymerase family B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase family B, exonuclease domain / Domain of unknown function (DUF1744) / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B / DNA polymerase alpha/delta/epsilon, subunit B / Ribonuclease H-like superfamily / DNA polymerase epsilon, subunit B / DNA polymerase epsilon catalytic subunit / Ribonuclease H superfamily / DNA polymerase family B, C-terminal domain / DNA polymerase alpha/epsilon subunit B
DNA polymerase epsilon catalytic subunit A / DNA polymerase epsilon subunit B
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsGoswami, P. / Purkiss, A. / Cheung, A. / Costa, A.
Funding supportUnited Kingdom , 3件
OrganizationGrant numberCountry
Wellcome TrustUnited Kingdom
Medical Research Council (United Kingdom)United Kingdom
Cancer Research UKUnited Kingdom
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 10, 2018 / Release: Dec 12, 2018
RevisionDateData content typeProviderType
1.0Dec 12, 2018Structure modelrepositoryInitial release

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Assembly

Deposited unit
B: DNA polymerase epsilon subunit B
A: DNA polymerase epsilon catalytic subunit A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,5244
Polymers183,3942
Non-polymers1312
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4430 Å2
ΔGint-24 kcal/mol
Surface area67970 Å2
MethodPISA

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Components

#1: Protein/peptide DNA polymerase epsilon subunit B / / DNA polymerase II subunit 2


Mass: 78408.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: DPB2, YPR175W, P9705.7 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P24482, EC: 2.7.7.7
#2: Protein/peptide DNA polymerase epsilon catalytic subunit A / DNA polymerase II subunit A


Mass: 104984.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: POL2, DUN2, YNL262W, N0825 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P21951, EC: 2.7.7.7
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2)
Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Image processingDetails: Volta phase plate
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161376 / Symmetry type: POINT

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