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- PDB-8ppu: Pyrococcus abyssi DNA polymerase D (PolD) in its editing mode bou... -

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Basic information

Entry
Database: PDB / ID: 8ppu
TitlePyrococcus abyssi DNA polymerase D (PolD) in its editing mode bound to a primer/template substrate containing three consecutive mismatches
Components
  • (DNA polymerase ...) x 2
  • DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')
  • DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*(GS)P*(C7R)P*(PST)P*(PST)P*(PST))-3')
  • DP2
KeywordsDNA BINDING PROTEIN / Polymerase / DNA / Replication / PolD / Archaea / Editing / Proofreading / Exonuclease / Nuclease
Function / homology
Function and homology information


exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / 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 small subunit, archaeal / DNA polymerase delta/II small subunit family / 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 / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / : ...DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / 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 / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA polymerase sliding clamp / DNA polymerase II small subunit
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsBetancurt-Anzola, L. / Martinez-Carranza, M. / Zatopek, K.M. / Gardner, A.F. / Sauguet, L.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR) France
CitationJournal: Nat Commun / Year: 2023
Title: Molecular basis for proofreading by the unique exonuclease domain of Family-D DNA polymerases.
Authors: Leonardo Betancurt-Anzola / Markel Martínez-Carranza / Marc Delarue / Kelly M Zatopek / Andrew F Gardner / Ludovic Sauguet /
Abstract: Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. ...Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. Family D replicative DNA polymerases (PolD), found exclusively in Archaea, contain an unusual RNA polymerase-like catalytic core, and a unique Mre11-like proofreading active site. Here, we present cryo-EM structures of PolD trapped in a proofreading mode, revealing an unanticipated correction mechanism that extends the repertoire of protein domains known to be involved in DNA proofreading. Based on our experimental structures, mutants of PolD were designed and their contribution to mismatch bypass and exonuclease kinetics was determined. This study sheds light on the convergent evolution of structurally distinct families of DNA polymerases, and the domain acquisition and exchange mechanism that occurred during the evolution of the replisome in the three domains of life.
History
DepositionJul 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation / citation_author
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
P: DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*(GS)P*(C7R)P*(PST)P*(PST)P*(PST))-3')
T: DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')
A: DNA polymerase II small subunit
C: DNA polymerase sliding clamp
D: DNA polymerase sliding clamp
E: DNA polymerase sliding clamp
B: DP2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)321,34413
Polymers321,0747
Non-polymers2696
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17060 Å2
ΔGint-103 kcal/mol
Surface area104460 Å2

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Components

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DNA chain , 2 types, 2 molecules PT

#1: DNA chain DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*(GS)P*(C7R)P*(PST)P*(PST)P*(PST))-3')


Mass: 6512.451 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pyrococcus abyssi GE5 (archaea)
#2: DNA chain DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')


Mass: 7717.934 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pyrococcus abyssi GE5 (archaea)

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

#3: Protein DNA polymerase II small subunit /


Mass: 74009.742 Da / Num. of mol.: 1 / Mutation: H451A
Source method: isolated from a genetically manipulated source
Details: DP1 subunit / Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: polB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V2F3
#4: Protein DNA polymerase sliding clamp / DNA clamp


Mass: 29471.764 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: PCNA / Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: pcn / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9UYX8

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Protein , 1 types, 1 molecules B

#5: Protein DP2


Mass: 144418.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Production host: Escherichia coli BL21(DE3) (bacteria)

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Non-polymers , 2 types, 6 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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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: Binary complex of PolD bound to PCNA and a primer/template duplex containing three consecutive mismatches
Type: COMPLEX / Entity ID: #3, #5, #4, #1-#2 / Source: RECOMBINANT
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
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 306575 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320099
ELECTRON MICROSCOPYf_angle_d0.42927289
ELECTRON MICROSCOPYf_dihedral_angle_d11.9292892
ELECTRON MICROSCOPYf_chiral_restr0.0413056
ELECTRON MICROSCOPYf_plane_restr0.0033393

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