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- PDB-6wjv: Structure of the Saccharomyces cerevisiae polymerase epsilon holo... -

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Basic information

Entry
Database: PDB / ID: 6wjv
TitleStructure of the Saccharomyces cerevisiae polymerase epsilon holoenzyme
Components
  • DNA polymerase epsilon catalytic subunit A
  • DNA polymerase epsilon subunit B
  • DNA polymerase epsilon subunit C
  • DNA polymerase epsilon subunit D
KeywordsCELL CYCLE / DNA replication / DNA polymerase
Function / homology
Function and homology information


: / : / chromatin remodeling => GO:0006338 / CHRAC / DNA-templated DNA replication maintenance of fidelity / gene conversion / : / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling ...: / : / chromatin remodeling => GO:0006338 / CHRAC / 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 / nucleosomal DNA binding / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / error-prone translesion synthesis / heterochromatin formation / base-excision repair, gap-filling / replication fork / base-excision repair / DNA-templated DNA replication / chromatin DNA binding / 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 / protein heterodimerization activity / nucleotide binding / mRNA binding / DNA damage response / DNA binding / zinc ion binding / nucleus / cytoplasm
Similarity search - Function
DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain ...DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Histone-fold / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase epsilon catalytic subunit A / DNA polymerase epsilon subunit B / DNA polymerase epsilon subunit C / DNA polymerase epsilon subunit D
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYuan, Z. / Georgescu, R. / Schauer, G.D. / O'Donnell, M. / Li, H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM131754 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM115809 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome.
Authors: Zuanning Yuan / Roxana Georgescu / Grant D Schauer / Michael E O'Donnell / Huilin Li /
Abstract: The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal ...The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ε revealing that the Dpb3-Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.
History
DepositionApr 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: DNA polymerase epsilon catalytic subunit A
2: DNA polymerase epsilon subunit B
3: DNA polymerase epsilon subunit C
4: DNA polymerase epsilon subunit D
hetero molecules


Theoretical massNumber of molelcules
Total (without water)379,2075
Polymers379,1424
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16190 Å2
ΔGint-129 kcal/mol
Surface area119230 Å2

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Components

#1: Protein DNA polymerase epsilon catalytic subunit A / 3'-5' exodeoxyribonuclease / DNA polymerase II subunit A


Mass: 255992.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL2, DUN2, YNL262W, N0825 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters
#2: Protein DNA polymerase epsilon subunit B / / DNA polymerase II subunit 2


Mass: 78425.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DPB2, YPR175W, P9705.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24482
#3: Protein DNA polymerase epsilon subunit C / / DNA polymerase II subunit C


Mass: 22694.014 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DPB3, YBR278W, YBR2015 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P27344
#4: Protein DNA polymerase epsilon subunit D / / DNA polymerase II subunit D


Mass: 22029.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DPB4, YDR121W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04603
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: DNA polymerase epsilon / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pRS
Buffer solutionpH: 7.5
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: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187298 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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