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- PDB-6t8h: Cryo-EM structure of the DNA-bound PolD-PCNA processive complex f... -

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Basic information

Entry
Database: PDB / ID: 6t8h
TitleCryo-EM structure of the DNA-bound PolD-PCNA processive complex from P. abyssi
Components
  • (DNA polymerase ...) x 2
  • DNA primerPrimer (molecular biology)
  • DNA template
  • DP2 subunit of D-family DNA-polymerase
KeywordsREPLICATION / PCNA / DP2 / PolD / DP1 / PIP-box
Function / homology
Function and homology information


exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intein-mediated protein splicing / DNA catabolic process / DNA polymerase processivity factor activity / regulation of DNA replication / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
Similarity search - Function
DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Intein splicing domain / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / DNA polymerase alpha/epsilon subunit B ...DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Intein splicing domain / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / DNA polymerase alpha/epsilon subunit B / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Hint domain superfamily / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / : / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Nucleic acid-binding, OB-fold / Alpha Beta
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA polymerase sliding clamp / DNA polymerase II small subunit / DNA polymerase II large subunit
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsMadru, C. / Raia, P. / Hugonneau Beaufet, I. / Pehau-Arnaudet, G. / England, P. / Lindhal, E. / Delarue, M. / Carroni, M. / Sauguet, L.
Funding support France, Sweden, 3items
OrganizationGrant numberCountry
French National Research AgencyANR-17-CE11-0005-01 France
Knut and Alice Wallenberg FoundationKAW 2015.0234 Sweden
Swedish Research Council2017-04641 Sweden
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA.
Authors: Clément Madru / Ghislaine Henneke / Pierre Raia / Inès Hugonneau-Beaufet / Gérard Pehau-Arnaudet / Patrick England / Erik Lindahl / Marc Delarue / Marta Carroni / Ludovic Sauguet /
Abstract: Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced ...Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.
History
DepositionOct 24, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 4, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 1, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Apr 8, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
E: DNA polymerase sliding clamp
A: DNA polymerase II small subunit
B: DP2 subunit of D-family DNA-polymerase
C: DNA polymerase sliding clamp
D: DNA polymerase sliding clamp
P: DNA primer
T: DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)316,14012
Polymers315,8237
Non-polymers3175
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14000 Å2
ΔGint-90 kcal/mol
Surface area99700 Å2
MethodPISA

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Components

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DNA polymerase ... , 2 types, 4 molecules ECDA

#1: Protein DNA polymerase sliding clamp / DNA clamp / Proliferating cell nuclear antigen homolog / PCNA


Mass: 29471.764 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (strain GE5 / Orsay) (archaea)
Strain: GE5 / Orsay / Gene: pcn, PYRAB13790, PAB1465 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UYX8
#2: Protein DNA polymerase II small subunit / / Pol II / Exodeoxyribonuclease small subunit


Mass: 69408.008 Da / Num. of mol.: 1 / Mutation: H451A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (strain GE5 / Orsay) (archaea)
Strain: GE5 / Orsay / Gene: polB, PYRAB01210, PAB2266 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9V2F3, DNA-directed DNA polymerase, exodeoxyribonuclease I

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Protein , 1 types, 1 molecules B

#3: Protein DP2 subunit of D-family DNA-polymerase


Mass: 144762.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Production host: Escherichia coli (E. coli)
References: UniProt: Q9V2F4*PLUS, DNA-directed DNA polymerase

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DNA chain , 2 types, 2 molecules PT

#4: DNA chain DNA primer / Primer (molecular biology)


Mass: 5519.542 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA template


Mass: 7717.934 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 5 molecules

#6: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe
#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1DNA-bound PolD-PCNA processive complex from P. abyssiCOMPLEX#1, #4-#50MULTIPLE SOURCES
2PolD-PCNA processive complexCOMPLEX#1-#31RECOMBINANT
3DNACOMPLEX#4-#51RECOMBINANT
Molecular weightValue: 310 kDa/nm / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pyrococcus abyssi (archaea)29292
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 8
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 5mA current / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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 / Nominal defocus min: 500 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4602
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1ScipionDiocletianparticle selection
2EPU2.0.3.79RELimage acquisition
4GctfCTF correction
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Indicated in the M&M
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149200 / Symmetry type: POINT

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