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- PDB-7nv0: Human Pol Kappa holoenzyme with wt PCNA -

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Basic information

Entry
Database: PDB / ID: 7nv0
TitleHuman Pol Kappa holoenzyme with wt PCNA
Components
  • DNA PrimerPrimer (molecular biology)
  • DNA Template
  • DNA polymerase kappaPOLK
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / 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 / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / MutLalpha complex binding / nucleotide-excision repair, DNA gap filling / Polymerase switching ...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 / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / MutLalpha complex binding / nucleotide-excision repair, DNA gap filling / 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) / 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 / replisome / response to L-glutamate / histone acetyltransferase binding / leading strand elongation / DNA polymerase processivity factor activity / G1/S-Specific Transcription / replication fork processing / response to dexamethasone / nuclear replication fork / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / cyclin-dependent protein kinase holoenzyme complex / mismatch repair / translesion synthesis / error-prone translesion synthesis / 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 / 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 replication / chromosome, telomeric region / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nuclear body / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / protein-containing complex binding / negative regulation of transcription by RNA polymerase II / enzyme binding / extracellular exosome / nucleoplasm / identical protein binding / metal ion binding / nucleus
Similarity search - Function
Rad18-like CCHC zinc finger / Proliferating cell nuclear antigen signature 2. / DNA polymerase type-Y, HhH motif / IMS family HHH motif / DNA polymerase IV / 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 ...Rad18-like CCHC zinc finger / Proliferating cell nuclear antigen signature 2. / DNA polymerase type-Y, HhH motif / IMS family HHH motif / DNA polymerase IV / 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 / Rad18, zinc finger UBZ4-type / Zinc finger UBZ4-type profile. / : / DNA polymerase, Y-family, little finger domain / impB/mucB/samB family C-terminal domain / UmuC domain / DNA polymerase, Y-family, little finger domain superfamily / impB/mucB/samB family / UmuC domain profile. / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Proliferating cell nuclear antigen / DNA polymerase kappa
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLancey, C. / De Biasio, A. / Hamdan, S.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: 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
DepositionMar 15, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 10, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: DNA polymerase kappa
B: Proliferating cell nuclear antigen
C: Proliferating cell nuclear antigen
D: Proliferating cell nuclear antigen
P: DNA Primer
T: DNA Template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)206,0437
Polymers205,5606
Non-polymers4821
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12700 Å2
ΔGint-66 kcal/mol
Surface area61490 Å2
MethodPISA

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Components

#1: Protein DNA polymerase kappa / POLK / DINB protein / DINP


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
#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
#3: DNA chain DNA Primer / Primer (molecular biology)


Mass: 7665.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA Template


Mass: 11677.539 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Pol kappa holoenzyme with wt-PCNACOMPLEX#1-#40MULTIPLE SOURCES
2Pol kappa holoenzymeCOMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41RECOMBINANT
Molecular weightValue: 0.206 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2100 mMPotassium acetateCH3COOK1
310 mMCalcium chlorideCaCl21
40.02 %NP-40H(C2H4O)nO(C6H4)C9H191
50.4 mMBiotinC10H16N2O3S1
61 mMDithiothreitolC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 40 mA / 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 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: 5 sec. / Electron dose: 47 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11114 / Details: Super resolution mode & AFIS used
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFINDCTF correction
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 12741853
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 254040 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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