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- PDB-9hzg: Ku70/80 bound to WRN-exo -

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Basic information

Entry
Database: PDB / ID: 9hzg
TitleKu70/80 bound to WRN-exo
Components
  • (DNA) x 2
  • (X-ray repair cross-complementing protein ...) x 2
  • Bifunctional 3'-5' exonuclease/ATP-dependent helicase WRN
KeywordsDNA BINDING PROTEIN / WRN / Ku70 / Ku80 / NHEJ
Function / homology
Function and homology information


positive regulation of hydrolase activity / 3'-flap-structured DNA binding / positive regulation of strand invasion / forked DNA-dependent helicase activity / telomeric G-quadruplex DNA binding / 8-hydroxy-2'-deoxyguanosine DNA binding / telomeric D-loop binding / regulation of growth rate / Ku70:Ku80 complex / DNA geometric change ...positive regulation of hydrolase activity / 3'-flap-structured DNA binding / positive regulation of strand invasion / forked DNA-dependent helicase activity / telomeric G-quadruplex DNA binding / 8-hydroxy-2'-deoxyguanosine DNA binding / telomeric D-loop binding / regulation of growth rate / Ku70:Ku80 complex / DNA geometric change / negative regulation of t-circle formation / DNA end binding / telomere maintenance via semi-conservative replication / Y-form DNA binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / telomeric D-loop disassembly / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / four-way junction helicase activity / t-circle formation / G-quadruplex DNA binding / bubble DNA binding / regulation of smooth muscle cell proliferation / cellular response to X-ray / MutLalpha complex binding / double-strand break repair via classical nonhomologous end joining / nuclear telomere cap complex / Cytosolic sensors of pathogen-associated DNA / Impaired BRCA2 binding to PALB2 / protein localization to nucleolus / IRF3-mediated induction of type I IFN / Processive synthesis on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / response to UV-C / recombinational repair / regulation of telomere maintenance / protein localization to chromosome, telomeric region / U3 snoRNA binding / exonuclease activity / positive regulation of neurogenesis / cellular hyperosmotic salinity response / DNA metabolic process / HDR through Single Strand Annealing (SSA) / 2-LTR circle formation / Homologous DNA Pairing and Strand Exchange / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / hematopoietic stem cell proliferation / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / telomeric repeat DNA binding / DNA synthesis involved in DNA repair / Impaired BRCA2 binding to RAD51 / 3'-5' DNA helicase activity / DNA 3'-5' helicase / replication fork processing / replicative senescence / 5'-deoxyribose-5-phosphate lyase activity / Presynaptic phase of homologous DNA pairing and strand exchange / hematopoietic stem cell differentiation / ATP-dependent activity, acting on DNA / telomere maintenance via telomerase / mismatch repair / SUMOylation of DNA damage response and repair proteins / site of DNA damage / four-way junction DNA binding / 3'-5' exonuclease activity / activation of innate immune response / neurogenesis / telomere maintenance / cyclin binding / DNA-(apurinic or apyrimidinic site) lyase / cellular response to starvation / replication fork / cellular response to leukemia inhibitory factor / DNA helicase activity / determination of adult lifespan / Nonhomologous End-Joining (NHEJ) / small-subunit processome / cellular response to gamma radiation / protein-DNA complex / G2/M DNA damage checkpoint / base-excision repair / double-strand break repair via homologous recombination / double-strand break repair via nonhomologous end joining / HDR through Homologous Recombination (HRR) / enzyme activator activity / cellular senescence / manganese ion binding / double-strand break repair / chromosome / Processing of DNA double-strand break ends / response to oxidative stress / double-stranded DNA binding / scaffold protein binding / DNA recombination / secretory granule lumen
Similarity search - Function
Helicase Helix-turn-helix domain / Helix-turn-helix domain / RQC domain / RQC / RQC domain / Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily ...Helicase Helix-turn-helix domain / Helix-turn-helix domain / RQC domain / RQC / RQC domain / Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 N-terminal alpha/beta domain / Helicase and RNase D C-terminal / ATP-dependent DNA helicase RecQ, zinc-binding domain / RecQ zinc-binding / HRDC domain / HRDC domain / DNA helicase, ATP-dependent, RecQ type / HRDC domain profile. / Ku70/Ku80 beta-barrel domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / SPOC-like, C-terminal domain superfamily / SAP domain superfamily / HRDC domain superfamily / 3'-5' exonuclease / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / 3'-5' exonuclease / 3'-5' exonuclease domain / HRDC-like superfamily / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / von Willebrand factor A-like domain superfamily / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / DNA / DNA (> 10) / DNA repair protein Ku70 / DNA repair protein Ku80 / Bifunctional 3'-5' exonuclease/ATP-dependent helicase WRN
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsHardwick, S.W. / Zahid, S. / Chaplin, A.K. / Ropars, R. / Charbonnier, J.B.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0026 France
CitationJournal: Nat Commun / Year: 2026
Title: Structural basis of Ku-mediated activation of WRN exonuclease activity.
Authors: Sayma Zahid / Jeanne Chauvat / Ilaria Ceppi / Floriana Cappiello / Benedetta Perdichizzi / Philippe Frit / Dennis Gomez / Steven W Hardwick / Pierre Legrand / Julien Karazi / Sonia Baconnais ...Authors: Sayma Zahid / Jeanne Chauvat / Ilaria Ceppi / Floriana Cappiello / Benedetta Perdichizzi / Philippe Frit / Dennis Gomez / Steven W Hardwick / Pierre Legrand / Julien Karazi / Sonia Baconnais / Gérard Pehau-Arnaudet / Sébastien Britton / Jean-Baptiste Charbonnier / Amanda K Chaplin / Pietro Pichierri / Petr Cejka / Patrick Calsou / Virginie Ropars /
Abstract: Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the ...Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the deficiency of which presents clinically as Werner syndrome, causing premature aging and cancer predisposition. The main DNA double strand-break sensor Ku70/80 heterodimer (Ku) is a known partner of WRN, which stimulates its nuclease activity. However, the molecular basis of Ku-WRN interplay is currently unknown. Here, we present a high resolution cryo-EM structure of human Ku bound to DNA in complex with the N-terminal WRN exonuclease domain. This structure reveals multiple interaction sites between WRN and the Ku:DNA complex. The catalytic domain of WRN-exo engages with the DNA ends, stabilized by the vWA-like Ku80 domain interacting with the N-terminal APLF-like Ku binding motif (A-KBM) of WRN. Most surprisingly, we visualize the SAP domain of Ku70 stabilized within this complex, and we identify specific contacts mediating this interaction. These interactions are validated by assessing the impact of point mutations on either side of the Ku-WRN interfaces on exonuclease activity with purified recombinant proteins, and on live protein recruitment at biphoton laser-damaged nuclear sites. Finally, we show that disruption of WRN-Ku70 interaction results in aberrant resection of stalled replication forks. Together, we define the architecture of the Ku-WRN exonuclease domain interface and its impact on WRN exonuclease activity, recruitment and replication fork processing.
History
DepositionJan 13, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 20, 2026Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: X-ray repair cross-complementing protein 6
E: Bifunctional 3'-5' exonuclease/ATP-dependent helicase WRN
B: X-ray repair cross-complementing protein 5
O: DNA
P: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)326,5687
Polymers325,8845
Non-polymers6842
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

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X-ray repair cross-complementing protein ... , 2 types, 2 molecules AB

#1: Protein X-ray repair cross-complementing protein 6 / 5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP- ...5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 1 / ATP-dependent DNA helicase II 70 kDa subunit / CTC box-binding factor 75 kDa subunit / CTCBF / DNA repair protein XRCC6 / Lupus Ku autoantigen protein p70 / Ku70 / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 6


Mass: 69945.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P12956, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement, Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases
#3: Protein X-ray repair cross-complementing protein 5 / 86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase ...86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase II 80 kDa subunit / CTC box-binding factor 85 kDa subunit / CTCBF / DNA repair protein XRCC5 / Ku80 / Ku86 / Lupus Ku autoantigen protein p86 / Nuclear factor IV / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining)


Mass: 82812.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Protein , 1 types, 1 molecules E

#2: Protein Bifunctional 3'-5' exonuclease/ATP-dependent helicase WRN / DNA helicase / RecQ-like type 3 / RecQ protein-like 2 / Werner syndrome protein


Mass: 162677.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WRN, RECQ3, RECQL2
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q14191, Hydrolases; Acting on ester bonds, DNA 3'-5' helicase

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DNA chain , 2 types, 2 molecules OP

#4: DNA chain DNA


Mass: 5253.415 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain DNA


Mass: 5195.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

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: Ku70/80 bound to WRN exo and DNA / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 8
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 47.24 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 262908
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83379 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 130.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310844
ELECTRON MICROSCOPYf_angle_d0.487814872
ELECTRON MICROSCOPYf_chiral_restr0.03661723
ELECTRON MICROSCOPYf_plane_restr0.00241783
ELECTRON MICROSCOPYf_dihedral_angle_d20.45923928

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