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- EMDB-14078: human Lig1-DNA-PCNA complex reconstituted in absence of ATP -

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Basic information

Entry
Database: EMDB / ID: EMD-14078
Titlehuman Lig1-DNA-PCNA complex reconstituted in absence of ATP
Map data
Sample
  • Complex: Human Ligase holoenzyme without ATP present
    • Protein or peptide: DNA ligase 1
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: Oligo19ddC
    • DNA: Oligo32
  • DNA: Oligo13P
  • Ligand: ADENOSINE MONOPHOSPHATE
KeywordsDNA / Replication / Complex / Ligase / PCNA / Ligation / Okazaki fragment maturation
Function / homology
Function and homology information


Okazaki fragment processing involved in mitotic DNA replication / DNA ligase 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 / purine-specific mismatch base pair DNA N-glycosylase activity / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence ...Okazaki fragment processing involved in mitotic DNA replication / DNA ligase 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 / purine-specific mismatch base pair DNA N-glycosylase activity / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / nuclear lamina / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Processive synthesis on the lagging strand / MutLalpha complex binding / PCNA complex / Removal of the Flap Intermediate / Telomere C-strand (Lagging Strand) Synthesis / lagging strand elongation / 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 / replisome / response to L-glutamate / Processive synthesis on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / DNA biosynthetic process / response to dexamethasone / histone acetyltransferase binding / DNA polymerase processivity factor activity / G1/S-Specific Transcription / leading strand elongation / Early Phase of HIV Life Cycle / nuclear replication fork / replication fork processing / SUMOylation of DNA replication proteins / POLB-Dependent Long Patch Base Excision Repair / PCNA-Dependent Long Patch Base Excision Repair / anatomical structure morphogenesis / response to cadmium ion / translesion synthesis / estrous cycle / mismatch repair / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / epithelial cell differentiation / liver regeneration / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / positive regulation of DNA repair / positive regulation of DNA replication / Translesion synthesis by REV1 / nuclear estrogen receptor binding / replication fork / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / male germ cell nucleus / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / Recognition of DNA damage by PCNA-containing replication complex / base-excision repair / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / cellular response to xenobiotic stimulus / Dual Incision in GG-NER / 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 / DNA recombination / damaged DNA binding / chromosome, telomeric region / nuclear body / cell division / DNA repair / intracellular membrane-bounded organelle / chromatin binding / centrosome / chromatin / protein-containing complex binding / enzyme binding / negative regulation of transcription by RNA polymerase II / DNA binding / extracellular exosome / nucleoplasm / ATP binding / metal ion binding / identical protein 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 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 ...: / 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. / 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.58 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_14078.png

Downloads & links

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Map

FileDownload / File: emd_14078.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 154 pix.
= 128.59 Å		0.84 Å/pix.
x 143 pix.
= 119.405 Å		0.84 Å/pix.
x 154 pix.
= 128.59 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-12.284604 - 14.311522500000001
Average (Standard dev.)0.000000000017628 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin655353
Dimensions143154154
Spacing154143154
CellA: 128.59 Å / B: 119.405 Å / C: 128.59 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_14078_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_14078_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human Ligase holoenzyme without ATP present

EntireName: Human Ligase holoenzyme without ATP present
Components
  • Complex: Human Ligase holoenzyme without ATP present
    • Protein or peptide: DNA ligase 1
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: Oligo19ddC
    • DNA: Oligo32
  • DNA: Oligo13P
  • Ligand: ADENOSINE MONOPHOSPHATE

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Supramolecule #1: Human Ligase holoenzyme without ATP present

SupramoleculeName: Human Ligase holoenzyme without ATP present / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #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: 101.877102 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK EWNGVVSESD SPVKRPGRKA ARVLGSEGEE EDEALSPAKG QKPALDCSQ VSPPRPATSP ENNASLSDTS PMDSSPSGIP KRRTARKQLP KRTIQEVLEE QSEDEDREAK RKKEEEEEET P KESLTEAE ...String:
MQRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK EWNGVVSESD SPVKRPGRKA ARVLGSEGEE EDEALSPAKG QKPALDCSQ VSPPRPATSP ENNASLSDTS PMDSSPSGIP KRRTARKQLP KRTIQEVLEE QSEDEDREAK RKKEEEEEET P KESLTEAE VATEKEGEDG DQPTTPPKPL KTSKAETPTE SVSEPEVATK QELQEEEEQT KPPRRAPKTL SSFFTPRKPA VK KEVKEEE PGAPGKEGAA EGPLDPSGYN PAKNNYHPVE DACWKPGQKV PYLAVARTFE KIEEVSARLR MVETLSNLLR SVV ALSPPD LLPVLYLSLN HLGPPQQGLE LGVGDGVLLK AVAQATGRQL ESVRAEAAEK GDVGLVAENS RSTQRLMLPP PPLT ASGVF SKFRDIARLT GSASTAKKID IIKGLFVACR HSEARFIARS LSGRLRLGLA EQSVLAALSQ AVSLTPPGQE FPPAM VDAG KGKTAEARKT WLEEQGMILK QTFCEVPDLD RIIPVLLEHG LERLPEHCKL SPGIPLKPML AHPTRGISEV LKRFEE AAF TCEYKYDGQR AQIHALEGGE VKIFSRNQED NTGKYPDIIS RIPKIKLPSV TSFILDTEAV AWDREKKQIQ PFQVLTT RK RKEVDASEIQ VQVCLYAFDL IYLNGESLVR EPLSRRRQLL RENFVETEGE FVFATSLDTK DIEQIAEFLE QSVKDSCE G LMVKTLDVDA TYEIAKRSHN WLKLKKDYLD GVGDTLDLVV IGAYLGRGKR AGRYGGFLLA SYDEDSEELQ AICKLGTGF SDEELEEHHQ SLKALVLPSP RPYVRIDGAV IPDHWLDPSA VWEVKCADLS LSPIYPAARG LVDSDKGISL RFPRFIRVRE DKQPEQATT SAQVACLYRK QSQIQNQQGE DSGSDPEDTY

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 #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 #6: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 6 / 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
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 / Number real images: 2966 / 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
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

Particle selectionNumber selected: 415322
Startup modelType of model: OTHER / Details: Relion Ab initio model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 73886
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7qnz:
human Lig1-DNA-PCNA complex reconstituted in absence of ATP

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