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- PDB-9f29: Pyrococcus abyssi PolD in complex with Rpa2 winged-helix domain c... -

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Basic information

Entry
Database: PDB / ID: 9f29
TitlePyrococcus abyssi PolD in complex with Rpa2 winged-helix domain class 1 (composite map)
Components
  • DNA polymerase II large subunit
  • DNA polymerase II small subunit
  • RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
KeywordsREPLICATION / DNA Polymerase / Archaea / RPA
Function / homology
Function and homology information


exodeoxyribonuclease I / DNA polymerase complex / single-stranded DNA 3'-5' DNA exonuclease activity / DNA catabolic process / DNA strand elongation involved in DNA replication / DNA-templated 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, N-terminal / DNA polymerase II large subunit DP2, central domain / DNA polymerase II large subunit DP2, catalytic domain / DNA polymerase II small subunit, archaeal / : / DNA polymerase II small subunit, N-terminal domain ...DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / : / : / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2, central domain / DNA polymerase II large subunit DP2, catalytic domain / DNA polymerase II small subunit, archaeal / : / DNA polymerase II small subunit, N-terminal domain / Replication factor A protein-like / DNA polymerase delta/II small subunit family / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
: / DNA polymerase II large subunit / RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination / DNA polymerase II small subunit
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsMartinez-Carranza, M. / Sauguet, L.
Funding support France, European Union, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR) France
Fondation pour la Recherche Medicale (FRM) France
H2020 Marie Curie Actions of the European CommissionEuropean Union
CitationJournal: Nat Commun / Year: 2024
Title: Communication between DNA polymerases and Replication Protein A within the archaeal replisome.
Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J ...Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J Iñaki Guijarro / Ghislaine Henneke / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. ...Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.
History
DepositionApr 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
A: DNA polymerase II small subunit
B: DNA polymerase II large subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,9467
Polymers233,6943
Non-polymers2524
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 15264.856 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: PAB2165 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z1
#2: Protein DNA polymerase II small subunit


Mass: 74009.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: polB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V2F3
#3: Protein DNA polymerase II large subunit / Pol II / DP2 / Exodeoxyribonuclease large subunit


Mass: 144418.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: polC, PF0019 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P81409, DNA-directed DNA polymerase, exodeoxyribonuclease I
#4: Chemical ChemComp-FE / FE (III) ION


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


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
Has protein modificationN

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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: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.23 MDa / Experimental value: YES
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 4.4 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169361 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313806
ELECTRON MICROSCOPYf_angle_d0.46318665
ELECTRON MICROSCOPYf_dihedral_angle_d3.9191855
ELECTRON MICROSCOPYf_chiral_restr0.0442049
ELECTRON MICROSCOPYf_plane_restr0.0052408

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