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

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Basic information

Entry
Database: PDB / ID: 7qo1
Titlecomplex of DNA ligase I and FEN1 on PCNA and DNA
Components
  • DNA ligase 1
  • Flap endonuclease 1
  • Oligo13P
  • Oligo19ddC
  • Oligo32
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / DNA / 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 / flap endonuclease activity / positive regulation of sister chromatid cohesion / telomere maintenance via semi-conservative replication / DNA ligase activity / double-stranded DNA exodeoxyribonuclease activity / DNA ligase (ATP) / DNA ligase (ATP) activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding ...Okazaki fragment processing involved in mitotic DNA replication / flap endonuclease activity / positive regulation of sister chromatid cohesion / telomere maintenance via semi-conservative replication / DNA ligase activity / double-stranded DNA exodeoxyribonuclease activity / DNA ligase (ATP) / DNA ligase (ATP) activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / 5'-flap endonuclease activity / purine-specific mismatch base pair DNA N-glycosylase activity / nuclear lamina / UV protection / 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 / DNA replication, removal of RNA primer / lagging strand elongation / Removal of the Flap Intermediate / HDR through MMEJ (alt-NHEJ) / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / 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 / replisome / 5'-3' exonuclease activity / exonuclease activity / response to L-glutamate / DNA biosynthetic process / histone acetyltransferase binding / DNA polymerase processivity factor activity / leading strand elongation / Early Phase of HIV Life Cycle / G1/S-Specific Transcription / response to dexamethasone / replication fork processing / nuclear replication fork / SUMOylation of DNA replication proteins / POLB-Dependent Long Patch Base Excision Repair / anatomical structure morphogenesis / PCNA-Dependent Long Patch Base Excision Repair / translesion synthesis / mismatch repair / response to cadmium ion / estrous cycle / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / epithelial cell differentiation / male germ cell nucleus / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / liver regeneration / Translesion synthesis by POLI / replication fork / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / double-strand break repair via homologous recombination / Translesion Synthesis by POLH / base-excision repair / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / memory / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / RNA-DNA hybrid ribonuclease activity / cellular response to UV / cellular response to xenobiotic stimulus / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / manganese ion binding / heart development / double-stranded DNA binding / endonuclease activity / DNA recombination / damaged DNA binding / Hydrolases; Acting on ester bonds / chromosome, telomeric region / DNA replication / nuclear body / cell division / DNA repair / 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 ...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 AMP-binding site. / ATP-dependent DNA ligase signature 2. / 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. / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / 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 / PIN-like domain superfamily / : / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / DNA / DNA (> 10) / Proliferating cell nuclear antigen / DNA ligase 1 / Flap endonuclease 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsBlair, K. / Tehseen, M. / Raducanu, V.S. / Shahid, T. / Lancey, C. / Cruehet, R. / Hamdan, S. / De Biasio, A.
Funding support United Kingdom, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA ligase 1
B: Proliferating cell nuclear antigen
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Oligo19ddC
I: Oligo13P
J: Oligo32
Y: Flap endonuclease 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,1029
Polymers233,7548
Non-polymers3471
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 5 molecules ABFGY

#1: Protein DNA ligase 1 / DNA ligase I / Polydeoxyribonucleotide synthase [ATP] 1


Mass: 84248.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LIG1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P18858, DNA ligase (ATP)
#2: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


Mass: 29088.061 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
#6: Protein Flap endonuclease 1 / FEN-1 / DNase IV / Flap structure-specific endonuclease 1 / Maturation factor 1 / hFEN-1


Mass: 42617.039 Da / Num. of mol.: 1 / Mutation: D181A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FEN1, RAD2 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P39748, Hydrolases; Acting on ester bonds

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DNA chain , 3 types, 3 molecules HIJ

#3: DNA chain Oligo19ddC


Mass: 5802.744 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Oligo13P


Mass: 3976.599 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain Oligo32


Mass: 9845.345 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM

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Details

Has ligand of interestY

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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: complex of DNA ligase I and FEN1 on PCNA and DNA / Type: COMPLEX / Entity ID: #2, #6, #1, #3, #5 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidC8H18N2O4S1
2100 mMpotassium acetateCH3CO2K1
310 mMmagnesium chlorideMgCl21
40.5 mMtris(2-carboxyethyl)phosphineC9H15O6P1
50.1 mMATPC10H16N5O13P31
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was coated with graphene oxide prior to use.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 18 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
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99243 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00514535
ELECTRON MICROSCOPYf_angle_d0.77719943
ELECTRON MICROSCOPYf_dihedral_angle_d18.1112412
ELECTRON MICROSCOPYf_chiral_restr0.0472314
ELECTRON MICROSCOPYf_plane_restr0.0062359

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