+Open data
-Basic information
Entry | Database: PDB / ID: 7nv1 | ||||||
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Title | Human Pol Kappa holoenzyme with Ub-PCNA | ||||||
Components |
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Keywords | REPLICATION / Translesion synthesis / TLS | ||||||
Function / homology | Function and homology information 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 / nuclear lamina / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis ...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 / nuclear lamina / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / 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 / replisome / nucleotide-excision repair, DNA gap filling / response to L-glutamate / histone acetyltransferase binding / DNA polymerase processivity factor activity / positive regulation of DNA replication / G1/S-Specific Transcription / replication fork processing / response to dexamethasone / leading strand elongation / nuclear replication fork / cyclin-dependent protein kinase holoenzyme complex / SUMOylation of DNA replication proteins / estrous cycle / epithelial cell differentiation / PCNA-Dependent Long Patch Base Excision Repair / positive regulation of DNA repair / mismatch repair / male germ cell nucleus / error-prone translesion synthesis / translesion synthesis / response to cadmium ion / DNA polymerase binding / liver regeneration / Translesion synthesis by REV1 / Translesion synthesis by POLK / base-excision repair, gap-filling / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / replication fork / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / 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 xenobiotic stimulus / cellular response to UV / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / chromosome, telomeric region / DNA replication / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nuclear body / DNA repair / chromatin binding / centrosome / DNA damage response / chromatin / protein-containing complex binding / enzyme binding / negative regulation of transcription by RNA polymerase II / extracellular exosome / nucleoplasm / identical protein binding / nucleus / metal ion binding Similarity search - Function | ||||||
Biological species | Homo sapiens (human) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.4 Å | ||||||
Authors | Lancey, C. / De Biasio, A. / Hamdan, S.M. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Nat Commun / Year: 2021 Title: Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA. Authors: Claudia Lancey / Muhammad Tehseen / Souvika Bakshi / Matthew Percival / Masateru Takahashi / Mohamed A Sobhy / Vlad S Raducanu / Kerry Blair / Frederick W Muskett / Timothy J Ragan / Ramon ...Authors: Claudia Lancey / Muhammad Tehseen / Souvika Bakshi / Matthew Percival / Masateru Takahashi / Mohamed A Sobhy / Vlad S Raducanu / Kerry Blair / Frederick W Muskett / Timothy J Ragan / Ramon Crehuet / Samir M Hamdan / Alfredo De Biasio / Abstract: Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the ...Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7nv1.cif.gz | 245.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7nv1.ent.gz | 184.6 KB | Display | PDB format |
PDBx/mmJSON format | 7nv1.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7nv1_validation.pdf.gz | 947.9 KB | Display | wwPDB validaton report |
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Full document | 7nv1_full_validation.pdf.gz | 961.6 KB | Display | |
Data in XML | 7nv1_validation.xml.gz | 42.2 KB | Display | |
Data in CIF | 7nv1_validation.cif.gz | 64.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nv/7nv1 ftp://data.pdbj.org/pub/pdb/validation_reports/nv/7nv1 | HTTPS FTP |
-Related structure data
Related structure data | 12602MC 7nv0C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10934 (Title: Cryo-EM multiframe micrographs of human Pol kappa complexed with DNA and monoubiquitylated PCNA Data size: 1.9 TB Data #1: Multiframe micrographs of human Pol κ complexed with monoubiquitylated PCNA and DNA [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 #2: Protein | | Mass: 98952.695 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLK, DINB1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9UBT6, DNA-directed DNA polymerase #3: DNA chain | | Mass: 7665.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #4: DNA chain | | Mass: 11677.539 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #5: Chemical | ChemComp-TTP / | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight | Value: 0.232 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Details: 40 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K |
Image recording | Average exposure time: 5 sec. / Electron dose: 47 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3889 / Details: Super resolution mode & AFIS used |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 11520 / Height: 8184 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 5275717 | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25234 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT |