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- EMDB-14080: complex of DNA ligase I and FEN1 on PCNA and DNA -

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Entry
Database: EMDB / ID: EMD-14080
Titlecomplex of DNA ligase I and FEN1 on PCNA and DNA
Map data
Sample
  • Complex: complex of DNA ligase I and FEN1 on PCNA and DNA
    • Protein or peptide: Proliferating cell nuclear antigen
    • Protein or peptide: Flap endonuclease 1
    • Protein or peptide: DNA ligase 1
    • DNA: Oligo19ddC
    • DNA: Oligo32
  • DNA: Oligo13P
  • Ligand: ADENOSINE MONOPHOSPHATE
KeywordsDNA / Replication / Complex / Ligase / PCNA / Ligation / FEN1 / Flap Endonuclease I / Okazaki fragment maturation
Function / homology
Function and homology information


Okazaki fragment processing involved in mitotic DNA replication / positive regulation of sister chromatid cohesion / flap endonuclease activity / telomere maintenance via semi-conservative replication / DNA ligase activity / double-stranded DNA exodeoxyribonuclease activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / DNA ligase (ATP) ...Okazaki fragment processing involved in mitotic DNA replication / positive regulation of sister chromatid cohesion / flap endonuclease activity / telomere maintenance via semi-conservative replication / DNA ligase activity / double-stranded DNA exodeoxyribonuclease activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / DNA ligase (ATP) / mitotic telomere maintenance via semi-conservative replication / DNA ligase (ATP) activity / nucleic acid metabolic process / purine-specific mismatch base pair DNA N-glycosylase activity / 5'-flap endonuclease activity / nuclear lamina / MutLalpha complex binding / DNA replication, removal of RNA primer / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / 5'-3' exonuclease activity / Processive synthesis on the lagging strand / UV protection / DNA ligation / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Polymerase switching on the C-strand of the telomere / 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 / HDR through MMEJ (alt-NHEJ) / lagging strand elongation / replisome / exonuclease activity / response to L-glutamate / histone acetyltransferase binding / DNA polymerase processivity factor activity / DNA biosynthetic process / G1/S-Specific Transcription / leading strand elongation / response to dexamethasone / replication fork processing / Early Phase of HIV Life Cycle / nuclear replication fork / SUMOylation of DNA replication proteins / POLB-Dependent Long Patch Base Excision Repair / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / cyclin-dependent protein kinase holoenzyme complex / anatomical structure morphogenesis / mismatch repair / translesion synthesis / response to cadmium ion / DNA polymerase binding / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / base-excision repair, gap-filling / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / replication fork / positive regulation of DNA replication / male germ cell nucleus / liver regeneration / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / double-strand break repair via homologous recombination / base-excision repair / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / receptor tyrosine kinase binding / memory / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / double-strand break repair / RNA-DNA hybrid ribonuclease activity / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / manganese ion binding / double-stranded DNA binding / DNA recombination / endonuclease activity / DNA replication / chromosome, telomeric region / damaged DNA binding / Hydrolases; Acting on ester bonds / nuclear body / cell division / intracellular membrane-bounded organelle
Similarity search - Function
Flap endonuclease 1 / XPG protein signature 2. / XPG conserved site / XPG protein signature 1. / XPG/Rad2 endonuclease / XPG, N-terminal / XPG-I domain / XPG N-terminal domain / XPG I-region / Xeroderma pigmentosum G I-region ...Flap endonuclease 1 / XPG protein signature 2. / XPG conserved site / XPG protein signature 1. / XPG/Rad2 endonuclease / XPG, N-terminal / XPG-I domain / XPG N-terminal domain / XPG I-region / Xeroderma pigmentosum G I-region / Xeroderma pigmentosum G N-region / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / ATP-dependent DNA ligase signature 2. / ATP-dependent DNA ligase AMP-binding site. / DNA ligase, ATP-dependent, C-terminal / ATP dependent DNA ligase C terminal region / DNA ligase, ATP-dependent, conserved site / ATP-dependent DNA ligase family profile. / DNA ligase, ATP-dependent, central / ATP dependent DNA ligase domain / 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 / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / PIN-like domain superfamily / : / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Proliferating cell nuclear antigen / DNA ligase 1 / Flap endonuclease 1
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsBlair K / Tehseen M / Raducanu VS / Shahid T / Lancey C / Cruehet R / Hamdan S / De Biasio A
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing.
Authors: Kerry Blair / Muhammad Tehseen / Vlad-Stefan Raducanu / Taha Shahid / Claudia Lancey / Fahad Rashid / Ramon Crehuet / Samir M Hamdan / Alfredo De Biasio /
Abstract: During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present ...During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIP) and DNA binding domain (PIP). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIP is released from PCNA and only PIP is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.
History
DepositionDec 23, 2021-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14080.png

Downloads & links

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Map

FileDownload / File: emd_14080.map.gz / Format: CCP4 / Size: 65.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 258 pix.
= 215.43 Å		0.84 Å/pix.
x 258 pix.
= 215.43 Å		0.84 Å/pix.
x 258 pix.
= 215.43 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.018752286 - 0.038317807
Average (Standard dev.)0.00020885404 (±0.0014837426)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions258258258
Spacing258258258
CellA=B=C: 215.43 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_14080_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14080_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : complex of DNA ligase I and FEN1 on PCNA and DNA

EntireName: complex of DNA ligase I and FEN1 on PCNA and DNA
Components
  • Complex: complex of DNA ligase I and FEN1 on PCNA and DNA
    • Protein or peptide: Proliferating cell nuclear antigen
    • Protein or peptide: Flap endonuclease 1
    • Protein or peptide: DNA ligase 1
    • DNA: Oligo19ddC
    • DNA: Oligo32
  • DNA: Oligo13P
  • Ligand: ADENOSINE MONOPHOSPHATE

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Supramolecule #1: complex of DNA ligase I and FEN1 on PCNA and DNA

SupramoleculeName: complex of DNA ligase I and FEN1 on PCNA and DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #6, #1, #3, #5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA ligase 1

MacromoleculeName: DNA ligase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA ligase (ATP)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 84.248555 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: CKESLTEAEV ATEKEGEDGD QPTTPPKPLK TSKAETPTES VSEPEVATKQ ELQEEEEQTK PPRRAPKTLS SFFTPRKPAV KKEVKEEEP GAPGKEGAAE GPLDPSGYNP AKNNYHPVED ACWKPGQKVP YLAVARTFEK IEEVSARLRM VETLSNLLRS V VALSPPDL ...String:
CKESLTEAEV ATEKEGEDGD QPTTPPKPLK TSKAETPTES VSEPEVATKQ ELQEEEEQTK PPRRAPKTLS SFFTPRKPAV KKEVKEEEP GAPGKEGAAE GPLDPSGYNP AKNNYHPVED ACWKPGQKVP YLAVARTFEK IEEVSARLRM VETLSNLLRS V VALSPPDL LPVLYLSLNH LGPPQQGLEL GVGDGVLLKA VAQATGRQLE SVRAEAAEKG DVGLVAENSR STQRLMLPPP PL TASGVFS KFRDIARLTG SASTAKKIDI IKGLFVACRH SEARFIARSL SGRLRLGLAE QSVLAALSQA VSLTPPGQEF PPA MVDAGK GKTAEARKTW LEEQGMILKQ TFCEVPDLDR IIPVLLEHGL ERLPEHCKLS PGIPLKPMLA HPTRGISEVL KRFE EAAFT CEYKYDGQRA QIHALEGGEV KIFSRNQEDN TGKYPDIISR IPKIKLPSVT SFILDTEAVA WDREKKQIQP FQVLT TRKR KEVDASEIQV QVCLYAFDLI YLNGESLVRE PLSRRRQLLR ENFVETEGEF VFATSLDTKD IEQIAEFLEQ SVKDSC EGL MVKTLDVDAT YEIAKRSHNW LKLKKDYLDG VGDTLDLVVI GAYLGRGKRA GRYGGFLLAS YDEDSEELQA ICKLGTG FS DEELEEHHQS LKALVLPSPR PYVRIDGAVI PDHWLDPSAV WEVKCADLSL SPIYPAARGL VDSDKGISLR FPRFIRVR E DKQPEQATTS AQVACLYRKQ SQIQNQQGED SGSDPEDTY

UniProtKB: DNA ligase 1

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Macromolecule #2: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.088061 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD ...String:
GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD GVKFSASGEL GNGNIKLSQT SNVDKEEEAV TIEMNEPVQL TFALRYLNFF TKATPLSSTV TLSMSADVPL VV EYKIADM GHLKYYLAPK IEDEEGS

UniProtKB: Proliferating cell nuclear antigen

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Macromolecule #6: Flap endonuclease 1

MacromoleculeName: Flap endonuclease 1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.617039 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGIQGLAKLI ADVAPSAIRE NDIKSYFGRK VAIDASMSIY QFLIAVRQGG DVLQNEEGET TSHLMGMFYR TIRMMENGIK PVYVFDGKP PQLKSGELAK RSERRAEAEK QLQQAQAAGA EQEVEKFTKR LVKVTKQHND ECKHLLSLMG IPYLDAPSEA E ASCAALVK ...String:
MGIQGLAKLI ADVAPSAIRE NDIKSYFGRK VAIDASMSIY QFLIAVRQGG DVLQNEEGET TSHLMGMFYR TIRMMENGIK PVYVFDGKP PQLKSGELAK RSERRAEAEK QLQQAQAAGA EQEVEKFTKR LVKVTKQHND ECKHLLSLMG IPYLDAPSEA E ASCAALVK AGKVYAAATE DMACLTFGSP VLMRHLTASE AKKLPIQEFH LSRILQELGL NQEQFVDLCI LLGSDYCESI RG IGPKRAV DLIQKHKSIE EIVRRLDPNK YPVPENWLHK EAHQLFLEPE VLDPESVELK WSEPNEEELI KFMCGEKQFS EER IRSGVK RLSKSRQGST QGRLDDFFKV TGSLSSAKRK EPEPKGSTKK KAKTGAAGKF KRGK

UniProtKB: Flap endonuclease 1

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Macromolecule #3: Oligo19ddC

MacromoleculeName: Oligo19ddC / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.802744 KDa
SequenceString:
(DG)(DC)(DT)(DT)(DC)(DT)(DG)(DT)(DG)(DC) (DT)(DG)(DA)(DT)(DG)(DC)(DG)(DT)(DOC)

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Macromolecule #4: Oligo13P

MacromoleculeName: Oligo13P / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.976599 KDa
SequenceString:
(DG)(DT)(DC)(DG)(DG)(DA)(DC)(DT)(DG)(DA) (DA)(DC)(DC)

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Macromolecule #5: Oligo32

MacromoleculeName: Oligo32 / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.845345 KDa
SequenceString:
(DG)(DG)(DT)(DT)(DC)(DA)(DG)(DT)(DC)(DC) (DG)(DA)(DC)(DG)(DA)(DC)(DG)(DC)(DA)(DT) (DC)(DA)(DG)(DC)(DA)(DC)(DA)(DG)(DA) (DA)(DG)(DC)

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Macromolecule #7: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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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
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMCH3CO2Kpotassium acetate
10.0 mMMgCl2magnesium chloride
0.5 mMC9H15O6Ptris(2-carboxyethyl)phosphine
0.1 mMC10H16N5O13P3ATP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec.
Details: The grid was coated with graphene oxide prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 18.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Relion Ab initio model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99243
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7qo1:
complex of DNA ligase I and FEN1 on PCNA and DNA

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