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- EMDB-15921: Open conformation of the complex of DNA ligase I on PCNA and DNA ... -

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Basic information

Entry
Database: EMDB / ID: EMD-15921
TitleOpen conformation of the complex of DNA ligase I on PCNA and DNA in the presence of ATP
Map data
Sample
  • Complex: complex of DNA ligase I on PCNA and DNA in the presence of ATP
    • Protein or peptide: DNA ligase 1
    • Protein or peptide: Proliferating cell nuclear antigen
Function / homology
Function and homology information


Okazaki fragment processing involved in mitotic DNA replication / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / DNA ligase activity / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / DNA ligase (ATP) / purine-specific mismatch base pair DNA N-glycosylase activity / DNA ligase (ATP) activity / positive regulation of DNA-directed DNA polymerase activity ...Okazaki fragment processing involved in mitotic DNA replication / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / DNA ligase activity / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / DNA ligase (ATP) / purine-specific mismatch base pair DNA N-glycosylase activity / DNA ligase (ATP) 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 / PCNA complex / DNA ligation / Removal of the Flap Intermediate / Processive synthesis 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 / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / lagging strand elongation / replisome / response to L-glutamate / histone acetyltransferase binding / leading strand elongation / DNA polymerase processivity factor activity / G1/S-Specific Transcription / replication fork processing / DNA biosynthetic process / response to dexamethasone / nuclear replication fork / Early Phase of HIV Life Cycle / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / POLB-Dependent Long Patch Base Excision Repair / cyclin-dependent protein kinase holoenzyme complex / mismatch repair / translesion synthesis / anatomical structure morphogenesis / response to cadmium ion / DNA polymerase binding / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / Gap-filling DNA repair synthesis and ligation in GG-NER / Translesion synthesis by POLI / 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 / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / heart development / DNA recombination / chromosome, telomeric region / damaged DNA binding / nuclear body / cell division / DNA repair / intracellular membrane-bounded organelle / centrosome / chromatin binding / chromatin / protein-containing complex binding / negative regulation of transcription by RNA polymerase II / enzyme binding / mitochondrion / DNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 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
DepositionOct 5, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15921.png

Downloads & links

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Map

FileDownload / File: emd_15921.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.086 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.055926003 - 0.099904865
Average (Standard dev.)0.00026172405 (±0.0025145041)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 238.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #2

Fileemd_15921_additional_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_15921_additional_2.map
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Half map: #2

Fileemd_15921_half_map_1.map
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Half map: #1

Fileemd_15921_half_map_2.map
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Sample components

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Entire : complex of DNA ligase I on PCNA and DNA in the presence of ATP

EntireName: complex of DNA ligase I on PCNA and DNA in the presence of ATP
Components
  • Complex: complex of DNA ligase I on PCNA and DNA in the presence of ATP
    • Protein or peptide: DNA ligase 1
    • Protein or peptide: Proliferating cell nuclear antigen

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Supramolecule #1: complex of DNA ligase I on PCNA and DNA in the presence of ATP

SupramoleculeName: complex of DNA ligase I on PCNA and DNA in the presence of ATP
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
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: 29.720342 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: DPSGYNPAKN NYHPVEDACW KPGQKVPYLA VARTFEKIEE VSARLRMVET LSNLLRSVVA LSPPDLLPVL YLSLNHLGPP QQGLELGVG DGVLLKAVAQ ATGRQLESVR AEAAEKGDVG LVAENSRSTQ RLMLPPPPLT ASGVFSKFRD IARLTGSAST A KKIDIIKG ...String:
DPSGYNPAKN NYHPVEDACW KPGQKVPYLA VARTFEKIEE VSARLRMVET LSNLLRSVVA LSPPDLLPVL YLSLNHLGPP QQGLELGVG DGVLLKAVAQ ATGRQLESVR AEAAEKGDVG LVAENSRSTQ RLMLPPPPLT ASGVFSKFRD IARLTGSAST A KKIDIIKG LFVACRHSEA RFIARSLSGR LRLGLAEQSV LAALSQAVSL TPPGQEFPPA MVDAGKGKTA EARKTWLEEQ GM ILKQTFC EVPDLDRIIP VLLEHGLERL PEHCKLS

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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: 28.441504 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 I

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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 mMC10H16N5O13P3ATPAdenosine triphosphate
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
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
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
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 107550
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

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

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