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- PDB-9cl7: Cryo-EM structure of FAN1-PCNA-DNA in final state -

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Basic information

Entry
Database: PDB / ID: 9cl7
TitleCryo-EM structure of FAN1-PCNA-DNA in final state
Components
  • DNA (40-MER)
  • DNA (46-MER) with (CAG)2 extrusion
  • Fanconi-associated nuclease 1
  • Proliferating cell nuclear antigen
KeywordsDNA BINDING PROTEIN / nuclease / CAG expansion / DNA repair / Huntington's disease / PROTEIN BINDING
Function / homology
Function and homology information


flap-structured DNA binding / phosphodiesterase I / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / 5'-flap endonuclease activity / purine-specific mismatch base pair DNA N-glycosylase activity / nuclear lamina / positive regulation of DNA-directed DNA polymerase activity ...flap-structured DNA binding / phosphodiesterase I / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / 5'-flap endonuclease activity / purine-specific mismatch base pair DNA N-glycosylase activity / nuclear lamina / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / MutLalpha complex binding / Processive synthesis on the lagging strand / PCNA complex / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / ubiquitin-modified protein reader activity / Removal of the Flap Intermediate from the C-strand / Transcription of E2F targets under negative control by DREAM complex / replisome / response to L-glutamate / 5'-3' exonuclease activity / phosphodiesterase I activity / response to dexamethasone / histone acetyltransferase binding / DNA polymerase processivity factor activity / leading strand elongation / G1/S-Specific Transcription / nuclear replication fork / replication fork processing / SUMOylation of DNA replication proteins / PCNA-Dependent Long Patch Base Excision Repair / response to cadmium ion / translesion synthesis / interstrand cross-link repair / estrous cycle / mismatch repair / intercellular bridge / cyclin-dependent protein kinase holoenzyme complex / Translesion synthesis by REV1 / Translesion synthesis by POLK / base-excision repair, gap-filling / DNA polymerase binding / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / epithelial cell differentiation / liver regeneration / positive regulation of DNA repair / Fanconi Anemia Pathway / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Termination of translesion DNA synthesis / replication fork / positive regulation of DNA replication / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / male germ cell nucleus / double-strand break repair via homologous recombination / nucleotide-excision repair / Dual Incision in GG-NER / HDR through Homologous Recombination (HRR) / receptor tyrosine kinase binding / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to hydrogen peroxide / cellular response to xenobiotic stimulus / cellular response to UV / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / chromatin organization / damaged DNA binding / chromosome, telomeric region / nuclear body / cilium / DNA repair / centrosome / chromatin binding / chromatin / protein-containing complex binding / enzyme binding / magnesium ion binding / negative regulation of transcription by RNA polymerase II / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / nucleus / cytosol
Similarity search - Function
: / : / : / Fanconi-associated nuclease 1, SAP subdomain / Fanconi-associated nuclease 1, TPR domain / Fanconi-associated nuclease 1-like / : / FAN1, HTH domain / VRR-NUC domain / VRR-NUC domain ...: / : / : / Fanconi-associated nuclease 1, SAP subdomain / Fanconi-associated nuclease 1, TPR domain / Fanconi-associated nuclease 1-like / : / FAN1, HTH domain / VRR-NUC domain / VRR-NUC domain / VRR_NUC / Rad18-like CCHC zinc finger / tRNA endonuclease-like domain superfamily / Proliferating cell nuclear antigen signature 2. / 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 / Rad18, zinc finger UBZ4-type / Zinc finger UBZ4-type profile. / :
Similarity search - Domain/homology
DNA / DNA (> 10) / Proliferating cell nuclear antigen / Fanconi-associated nuclease 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.83 Å
AuthorsLi, F. / Pluciennik, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural and molecular basis of PCNA-activated FAN1 nuclease function in DNA repair.
Authors: F Li / A S Phadte / M Bhatia / S Barndt / A R Monte Carlo Iii / C-F D Hou / R Yang / S Strock / A Pluciennik /
Abstract: FAN1 is a DNA dependent nuclease whose proper function is essential for maintaining human health. For example, a genetic variant in FAN1, Arg507 to His hastens onset of Huntington's disease, a repeat ...FAN1 is a DNA dependent nuclease whose proper function is essential for maintaining human health. For example, a genetic variant in FAN1, Arg507 to His hastens onset of Huntington's disease, a repeat expansion disorder for which there is no cure. How the Arg507His mutation affects FAN1 structure and enzymatic function is unknown. Using cryo-EM and biochemistry, we have discovered that FAN1 arginine 507 is critical for its interaction with PCNA, and mutation of Arg507 to His attenuates assembly of the FAN1-PCNA complex on a disease-relevant extrahelical DNA extrusions formed within DNA repeats. This mutation concomitantly abolishes PCNA-FAN1-dependent cleavage of such extrusions, thus unraveling the molecular basis for a specific mutation in FAN1 that dramatically hastens the onset of Huntington's disease. These results underscore the importance of PCNA to the genome stabilizing function of FAN1.
History
DepositionJul 10, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
Revision 1.2Sep 3, 2025Group: Data collection / Database references / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA (46-MER) with (CAG)2 extrusion
F: DNA (40-MER)
B: Proliferating cell nuclear antigen
C: Proliferating cell nuclear antigen
D: Proliferating cell nuclear antigen
E: Fanconi-associated nuclease 1


Theoretical massNumber of molelcules
Total (without water)185,7326
Polymers185,7326
Non-polymers00
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: DNA chain DNA (46-MER) with (CAG)2 extrusion


Mass: 14231.137 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (40-MER)


Mass: 12256.890 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


Mass: 28795.752 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PCNA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P12004
#4: Protein Fanconi-associated nuclease 1 / FANCD2/FANCI-associated nuclease 1 / hFAN1 / Myotubularin-related protein 15


Mass: 72856.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FAN1, KIAA1018, MTMR15 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9Y2M0, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters, phosphodiesterase I
Has protein modificationY

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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: Ternary complex of FAN1-PCNA-DNA in final state / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313109
ELECTRON MICROSCOPYf_angle_d0.68318089
ELECTRON MICROSCOPYf_dihedral_angle_d21.6732344
ELECTRON MICROSCOPYf_chiral_restr0.042071
ELECTRON MICROSCOPYf_plane_restr0.0032018

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