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- EMDB-45568: Cryo-EM structure of FAN1, PCNA and DNA substrate with (CAG)2 ext... -

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Basic information

Entry
Database: EMDB / ID: EMD-45568
TitleCryo-EM structure of FAN1, PCNA and DNA substrate with (CAG)2 extrusion in intermediate state
Map data
Sample
  • Complex: Ternary complex of FAN1-PCNA-DNA in intermediate state
    • Protein or peptide: Proliferating cell nuclear antigen
    • Protein or peptide: Fanconi-associated nuclease 1
    • DNA: DNA (5'-D(P*CP*GP*AP*AP*TP*TP*TP*CP*TP*AP*GP*AP*CP*TP*CP*GP*AP*GP*AP*TP*CP*AP*G)-3')
    • DNA: DNA (5'-D(P*AP*TP*CP*TP*CP*GP*AP*GP*TP*CP*TP*AP*GP*AP*AP*AP*TP*TP*CP*G)-3')
Keywordsnuclease / CAG expansion / DNA repair / Huntington's disease / DNA BINDING PROTEIN
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 / MutLalpha complex binding / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / PCNA complex / Removal of the Flap Intermediate / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / 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) / Transcription of E2F targets under negative control by DREAM complex / Removal of the Flap Intermediate from the C-strand / ubiquitin-modified protein reader activity / 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 / estrous cycle / mismatch repair / interstrand cross-link repair / intercellular bridge / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / liver regeneration / epithelial cell differentiation / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / positive regulation of DNA replication / Gap-filling DNA repair synthesis and ligation in GG-NER / replication fork / nuclear estrogen receptor binding / male germ cell nucleus / nucleotide-excision repair / Fanconi Anemia Pathway / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / double-strand break repair via homologous recombination / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / cellular response to xenobiotic stimulus / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / 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
Proliferating cell nuclear antigen / Fanconi-associated nuclease 1
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsLi F / Pluciennik A
Funding support United States, 1 items
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
DepositionJun 28, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45568.png

Downloads & links

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Map

FileDownload / File: emd_45568.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.944 Å		1.07 Å/pix.
x 256 pix.
= 274.944 Å		1.07 Å/pix.
x 256 pix.
= 274.944 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.074 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.36328876 - 0.7505869
Average (Standard dev.)-0.0002658487 (±0.017525809)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.944 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_45568_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_45568_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of FAN1-PCNA-DNA in intermediate state

EntireName: Ternary complex of FAN1-PCNA-DNA in intermediate state
Components
  • Complex: Ternary complex of FAN1-PCNA-DNA in intermediate state
    • Protein or peptide: Proliferating cell nuclear antigen
    • Protein or peptide: Fanconi-associated nuclease 1
    • DNA: DNA (5'-D(P*CP*GP*AP*AP*TP*TP*TP*CP*TP*AP*GP*AP*CP*TP*CP*GP*AP*GP*AP*TP*CP*AP*G)-3')
    • DNA: DNA (5'-D(P*AP*TP*CP*TP*CP*GP*AP*GP*TP*CP*TP*AP*GP*AP*AP*AP*TP*TP*CP*G)-3')

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Supramolecule #1: Ternary complex of FAN1-PCNA-DNA in intermediate state

SupramoleculeName: Ternary complex of FAN1-PCNA-DNA in intermediate state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.795752 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MFEARLVQGS ILKKVLEALK DLINEACWDI SSSGVNLQSM DSSHVSLVQL TLRSEGFDTY RCDRNLAMGV NLTSMSKILK CAGNEDIIT LRAEDNADTL ALVFEAPNQE KVSDYEMKLM DLDVEQLGIP EQEYSCVVKM PSGEFARICR DLSHIGDAVV I SCAKDGVK ...String:
MFEARLVQGS ILKKVLEALK DLINEACWDI SSSGVNLQSM DSSHVSLVQL TLRSEGFDTY RCDRNLAMGV NLTSMSKILK CAGNEDIIT LRAEDNADTL ALVFEAPNQE KVSDYEMKLM DLDVEQLGIP EQEYSCVVKM PSGEFARICR DLSHIGDAVV I SCAKDGVK FSASGELGNG NIKLSQTSNV DKEEEAVTIE MNEPVQLTFA LRYLNFFTKA TPLSSTVTLS MSADVPLVVE YK IADMGHL KYYLAPKIED EEGS

UniProtKB: Proliferating cell nuclear antigen

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Macromolecule #2: Fanconi-associated nuclease 1

MacromoleculeName: Fanconi-associated nuclease 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.853436 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
HPYYLRSFLV VLKTVLENED DMLLFDEQEK GIVTKFYQLS ATGQKLYVRL FQRKLSWIKM TKLEYEEIAL DLTPVIEELT NAGFLQTES ELQELSEVLE LLSAPELKSL AKTFHLVNPN GQKQQLVDAF LKLAKQRSVC TWGKNKPGIG AVILKRAKAL A GQSVRICK GPRAVFSRIL LLFSL

UniProtKB: Fanconi-associated nuclease 1

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Macromolecule #3: DNA (5'-D(P*CP*GP*AP*AP*TP*TP*TP*CP*TP*AP*GP*AP*CP*TP*CP*GP*AP*GP...

MacromoleculeName: DNA (5'-D(P*CP*GP*AP*AP*TP*TP*TP*CP*TP*AP*GP*AP*CP*TP*CP*GP*AP*GP*AP*TP*CP*AP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.064585 KDa
SequenceString:
(DC)(DG)(DA)(DA)(DT)(DT)(DT)(DC)(DT)(DA) (DG)(DA)(DC)(DT)(DC)(DG)(DA)(DG)(DA)(DT) (DC)(DA)(DG)

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Macromolecule #4: DNA (5'-D(P*AP*TP*CP*TP*CP*GP*AP*GP*TP*CP*TP*AP*GP*AP*AP*AP*TP*TP...

MacromoleculeName: DNA (5'-D(P*AP*TP*CP*TP*CP*GP*AP*GP*TP*CP*TP*AP*GP*AP*AP*AP*TP*TP*CP*G)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.132991 KDa
SequenceString:
(DA)(DT)(DC)(DT)(DC)(DG)(DA)(DG)(DT)(DC) (DT)(DA)(DG)(DA)(DA)(DA)(DT)(DT)(DC)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 300000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-9cg4:
Cryo-EM structure of FAN1, PCNA and DNA substrate with (CAG)2 extrusion in intermediate state

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