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- PDB-8s8p: Restriction on Ku Inward Translocation Caps Telomere Ends -

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Basic information

Entry
Database: PDB / ID: 8s8p
TitleRestriction on Ku Inward Translocation Caps Telomere Ends
Components
  • ATP-dependent DNA helicase II subunit 1
  • ATP-dependent DNA helicase II subunit 2
  • DNA (5'-D(*AP*CP*AP*CP*AP*CP*AP*CP*AP*CP*CP*CP*AP*CP*AP*CP*AP*CP*CP*AP*C)-3')
  • DNA (5'-D(*GP*TP*GP*GP*TP*GP*TP*GP*TP*GP*GP*GP*TP*GP*TP*GP*TP*GP*TP*GP*T)-3')
  • DNA-binding protein RAP1
KeywordsDNA BINDING PROTEIN / Telomere / NHEJ / Rap1 / Ku / Chromosome / DNA Repair / Mutagenesis
Function / homology
Function and homology information


positive regulation of ribosomal protein gene transcription by RNA polymerase II / G-quadruplex DNA formation / donor selection / mitochondrial double-strand break repair via homologous recombination / telomeric G-quadruplex DNA binding / protein localization to chromosome / establishment of protein-containing complex localization to telomere / protection from non-homologous end joining at telomere / establishment of protein localization to telomere / Ku70:Ku80 complex ...positive regulation of ribosomal protein gene transcription by RNA polymerase II / G-quadruplex DNA formation / donor selection / mitochondrial double-strand break repair via homologous recombination / telomeric G-quadruplex DNA binding / protein localization to chromosome / establishment of protein-containing complex localization to telomere / protection from non-homologous end joining at telomere / establishment of protein localization to telomere / Ku70:Ku80 complex / establishment of protein localization to chromatin / telomere maintenance via telomere lengthening / shelterin complex / double-stranded telomeric DNA binding / G-quadruplex DNA binding / double-strand break repair via break-induced replication / telomerase RNA binding / recombinational repair / silent mating-type cassette heterochromatin formation / regulation of glycolytic process / DNA binding, bending / nucleosomal DNA binding / nuclear chromosome / telomeric DNA binding / TFIID-class transcription factor complex binding / subtelomeric heterochromatin formation / cis-regulatory region sequence-specific DNA binding / Neutrophil degranulation / telomere maintenance / DNA helicase activity / TBP-class protein binding / double-strand break repair via homologous recombination / helicase activity / protein-DNA complex / double-strand break repair via nonhomologous end joining / nuclear envelope / chromatin organization / histone binding / transcription regulator complex / damaged DNA binding / DNA helicase / sequence-specific DNA binding / RNA polymerase II-specific DNA-binding transcription factor binding / DNA-binding transcription factor activity, RNA polymerase II-specific / chromosome, telomeric region / DNA-binding transcription factor activity / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Rap1, DNA-binding domain / Rap1, DNA-binding / TRF2-interacting telomeric protein/Rap1, C-terminal / TRF2-interacting telomeric protein/Rap1, C-terminal domain superfamily / TRF2-interacting telomeric protein/Rap1 - C terminal domain / TE2IP/Rap1 / : / Ku70 / Ku80 / Ku70/Ku80 C-terminal arm ...Rap1, DNA-binding domain / Rap1, DNA-binding / TRF2-interacting telomeric protein/Rap1, C-terminal / TRF2-interacting telomeric protein/Rap1, C-terminal domain superfamily / TRF2-interacting telomeric protein/Rap1 - C terminal domain / TE2IP/Rap1 / : / Ku70 / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain / Ku70/Ku80 N-terminal alpha/beta 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 / BRCT domain / Myb-type HTH DNA-binding domain profile. / Myb domain / Myb-like DNA-binding domain / breast cancer carboxy-terminal domain / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / von Willebrand factor A-like domain superfamily / Homeobox-like domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-binding protein RAP1 / ATP-dependent DNA helicase II subunit 1 / ATP-dependent DNA helicase II subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsMattarocci, S. / Baconnais, S. / Roisne-Hamelin, F. / Pobiega, S. / Alibert, O. / Morin, V. / Deshayes, A. / Veaute, X. / Ropars, V. / Mazon, G. ...Mattarocci, S. / Baconnais, S. / Roisne-Hamelin, F. / Pobiega, S. / Alibert, O. / Morin, V. / Deshayes, A. / Veaute, X. / Ropars, V. / Mazon, G. / Busso, D. / Fernandez Varela, P. / Le Cam, E. / Charbonnier, J. / Cuniasse, P. / Marcand, S.
Funding support France, 5items
OrganizationGrant numberCountry
Fondation pour la Recherche Medicale (FRM)EQU202203014702 France
Agence Nationale de la Recherche (ANR)ANR-15CE12-0007 DNA-Life France
Fondation ARC France
La ligue contre le cancer France
French Alternative Energies and Atomic Energy Commission (CEA) France
CitationJournal: Nat Commun / Year: 2025
Title: Restriction of Ku translocation protects telomere ends.
Authors: Stefano Mattarocci / Sonia Baconnais / Florian Roisné-Hamelin / Sabrina Pobiega / Olivier Alibert / Vincent Morin / Alice Deshayes / Xavier Veaute / Virginie Ropars / Maelenn Chevreuil / ...Authors: Stefano Mattarocci / Sonia Baconnais / Florian Roisné-Hamelin / Sabrina Pobiega / Olivier Alibert / Vincent Morin / Alice Deshayes / Xavier Veaute / Virginie Ropars / Maelenn Chevreuil / Johannes Mehringer / Didier Busso / Gerard Mazon / Paloma Fernandez Varela / Éric Le Cam / Jean-Baptiste Charbonnier / Philippe Cuniasse / Stéphane Marcand /
Abstract: Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is ...Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, Rap1 in Saccharomyces cerevisiae. Here, we find that Rap1 directly inhibits Ku's NHEJ function at telomeres. A single Rap1 molecule near a double-stand break suppresses NHEJ without displacing Ku in cells. Furthermore, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM shows Rap1 blocks Ku's inward translocation on DNA - an essential step for NHEJ at DSBs. Nanopore sequencing of telomere fusions confirms this mechanism protects native telomere ends. These findings uncover a telomere protection mechanism where Rap1 restricts Ku's inward translocation. This switches Ku from a repair-promoting to a protective role preventing NHEJ at telomeres.
History
DepositionMar 7, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Aug 13, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA (5'-D(*AP*CP*AP*CP*AP*CP*AP*CP*AP*CP*CP*CP*AP*CP*AP*CP*AP*CP*CP*AP*C)-3')
D: DNA (5'-D(*GP*TP*GP*GP*TP*GP*TP*GP*TP*GP*GP*GP*TP*GP*TP*GP*TP*GP*TP*GP*T)-3')
K: ATP-dependent DNA helicase II subunit 1
L: ATP-dependent DNA helicase II subunit 2
R: DNA-binding protein RAP1


Theoretical massNumber of molelcules
Total (without water)173,2575
Polymers173,2575
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area30450 Å2
ΔGint-178 kcal/mol
Surface area75810 Å2
MethodPISA

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Components

#1: DNA chain DNA (5'-D(*AP*CP*AP*CP*AP*CP*AP*CP*AP*CP*CP*CP*AP*CP*AP*CP*AP*CP*CP*AP*C)-3')


Mass: 6244.084 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (5'-D(*GP*TP*GP*GP*TP*GP*TP*GP*TP*GP*GP*GP*TP*GP*TP*GP*TP*GP*TP*GP*T)-3')


Mass: 6643.248 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein ATP-dependent DNA helicase II subunit 1 / ATP-dependent DNA helicase II subunit Ku70 / High affinity DNA-binding factor subunit 1


Mass: 65531.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: YKU70, HDF1, NES24, YMR284W, YM8021.10 / Production host: Escherichia coli (E. coli) / References: UniProt: P32807, DNA helicase
#4: Protein ATP-dependent DNA helicase II subunit 2 / ATP-dependent DNA helicase II subunit Ku80 / High affinity DNA-binding factor subunit 2 / Yeast Ku80


Mass: 66469.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: YKU80, HDF2, YMR106C, YM9718.05C / Production host: Escherichia coli (E. coli) / References: UniProt: Q04437, DNA helicase
#5: Protein DNA-binding protein RAP1 / Repressor/activator site-binding protein / SBF-E / TUF


Mass: 28368.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAP1, GRF1, TUF1, YNL216W, N1310 / Production host: Escherichia coli (E. coli) / References: UniProt: P11938
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

Component
IDNameTypeEntity IDParent-IDSource
1Ternary Complex of Ku Rap1 and DNACOMPLEX#3-#4, #2, #1, #50MULTIPLE SOURCES
2ATP-dependent DNA helicase II and DNA-binding protein RAP1COMPLEX#3-#51RECOMBINANT
3Double stranded DNACOMPLEX#2, #11RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Saccharomyces cerevisiae (brewer's yeast)4932
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 8 / Details: Tris-HCL 10mM, Nacl 50 mM, pH 8.04
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 FIELD / Nominal defocus max: 750 nm / Nominal defocus min: 250 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selection
7NAMD2.14model fittingMDFF protocole
9PHENIX1.21model refinementRealSpaceRefinement of the final MDF model
13cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1544386
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 436644 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial Fitting of the model in the cryo-EM map was achieved using ChimeraX and this structure served as starting point for MDFF refinement (Flexible fitting) using NAMD 2.14.
Atomic model buildingDetails: Model build using PDB entries 5Y58 and 3UKG / Source name: Other / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212465
ELECTRON MICROSCOPYf_angle_d0.52317018
ELECTRON MICROSCOPYf_dihedral_angle_d16.4451938
ELECTRON MICROSCOPYf_chiral_restr0.0391884
ELECTRON MICROSCOPYf_plane_restr0.0042055

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