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- PDB-8b8t: Open conformation of the complex of DNA ligase I on PCNA and DNA ... -

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Open data


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Basic information

Entry
Database: PDB / ID: 8b8t
TitleOpen conformation of the complex of DNA ligase I on PCNA and DNA in the presence of ATP
Components
  • DNA ligase 1
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / DNA / Complex / Ligase / PCNA / Ligation / Okazaki fragment maturation
Function / homology
Function and homology information


Okazaki fragment processing involved in mitotic DNA replication / DNA ligase 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 / purine-specific mismatch base pair DNA N-glycosylase activity / positive regulation of DNA-directed DNA polymerase activity ...Okazaki fragment processing involved in mitotic DNA replication / DNA ligase 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 / purine-specific mismatch base pair DNA N-glycosylase activity / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / MutLalpha complex binding / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / 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 / lagging strand elongation / replisome / 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 / base-excision repair / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / receptor tyrosine kinase binding / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / DNA recombination / chromosome, telomeric region / damaged DNA binding / nuclear body / cell division / intracellular membrane-bounded organelle / DNA repair / centrosome / chromatin binding / protein-containing complex binding / chromatin / enzyme binding / negative regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding
Similarity search - Function
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 / 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 / : / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Proliferating cell nuclear antigen / DNA ligase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 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
DepositionOct 5, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA ligase 1
E: Proliferating cell nuclear antigen
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen


Theoretical massNumber of molelcules
Total (without water)115,0454
Polymers115,0454
Non-polymers00
Water00
1


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

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Components

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


Mass: 29720.342 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: 28441.504 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

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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 on PCNA and DNA in the presence of ATP
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
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
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: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107550 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047702
ELECTRON MICROSCOPYf_angle_d0.6210420
ELECTRON MICROSCOPYf_dihedral_angle_d4.0451060
ELECTRON MICROSCOPYf_chiral_restr0.0441242
ELECTRON MICROSCOPYf_plane_restr0.0041334

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