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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-12602 | |||||||||
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Title | Human Pol Kappa holoenzyme with Ub-PCNA | |||||||||
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![]() | Translesion synthesis / TLS / REPLICATION | |||||||||
Function / homology | ![]() 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 / G1/S-Specific Transcription / replication fork processing / response to dexamethasone / leading strand elongation / nuclear replication fork / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / mismatch repair / error-prone translesion synthesis / translesion synthesis / response to cadmium ion / DNA polymerase binding / cyclin-dependent protein kinase holoenzyme complex / epithelial cell differentiation / positive regulation of DNA repair / 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 / positive regulation of DNA replication / replication fork / male germ cell nucleus / nuclear estrogen receptor binding / liver regeneration / 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 / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / DNA replication / damaged DNA binding / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nuclear body / DNA repair / centrosome / DNA damage response / chromatin binding / protein-containing complex binding / chromatin / negative regulation of transcription by RNA polymerase II / enzyme binding / extracellular exosome / nucleoplasm / identical protein binding / nucleus / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.4 Å | |||||||||
![]() | Lancey C / De Biasio A | |||||||||
Funding support | ![]()
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![]() | ![]() 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
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 8.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.4 KB 20.4 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 13.8 KB | Display | ![]() |
Images | ![]() | 62.1 KB | ||
Filedesc metadata | ![]() | 7.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7nv1MC ![]() 7nv0C C: citing same article ( M: atomic model generated by this map |
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Similar structure data | |
EM raw data | ![]() Data size: 1.9 TB Data #1: Multiframe micrographs of human Pol κ complexed with monoubiquitylated PCNA and DNA [micrographs - multiframe]) |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.086 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Pol kappa holoenzyme with Ub-PCNA
Entire | Name: Pol kappa holoenzyme with Ub-PCNA |
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Components |
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-Supramolecule #1: Pol kappa holoenzyme with Ub-PCNA
Supramolecule | Name: Pol kappa holoenzyme with Ub-PCNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Molecular weight | Theoretical: 232 KDa |
-Supramolecule #2: Pol kappa holoenzyme
Supramolecule | Name: Pol kappa holoenzyme / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() |
-Supramolecule #3: DNA
Supramolecule | Name: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4 |
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Source (natural) | Organism: synthetic construct (others) |
-Macromolecule #1: Proliferating cell nuclear antigen
Macromolecule | Name: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 29.088061 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: DNA polymerase kappa
Macromolecule | Name: DNA polymerase kappa / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 98.952695 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MDSTKEKCDS YKDDLLLRMG LNDNKAGMEG LDKEKINKII MEATKGSRFY GNELKKEKQV NQRIENMMQQ KAQITSQQLR KAQLQVDRF AMELEQSRNL SNTIVHIDMD AFYAAVEMRD NPELKDKPIA VGSMSMLSTS NYHARRFGVR AAMPGFIAKR L CPQLIIVP ...String: MDSTKEKCDS YKDDLLLRMG LNDNKAGMEG LDKEKINKII MEATKGSRFY GNELKKEKQV NQRIENMMQQ KAQITSQQLR KAQLQVDRF AMELEQSRNL SNTIVHIDMD AFYAAVEMRD NPELKDKPIA VGSMSMLSTS NYHARRFGVR AAMPGFIAKR L CPQLIIVP PNFDKYRAVS KEVKEILADY DPNFMAMSLD EAYLNITKHL EERQNWPEDK RRYFIKMGSS VENDNPGKEV NK LSEHERS ISPLLFEESP SDVQPPGDPF QVNFEEQNNP QILQNSVVFG TSAQEVVKEI RFRIEQKTTL TASAGIAPNT MLA KVCSDK NKPNGQYQIL PNRQAVMDFI KDLPIRKVSG IGKVTEKMLK ALGIITCTEL YQQRALLSLL FSETSWHYFL HISL GLGST HLTRDGERKS MSVERTFSEI NKAEEQYSLC QELCSELAQD LQKERLKGRT VTIKLKNVNF EVKTRASTVS SVVST AEEI FAIAKELLKT EIDADFPHPL RLRLMGVRIS SFPNEEDRKH QQRSIIGFLQ AGNQALSATE CTLEKTDKDK FVKPLE MSH KKSFFDKKRS ERKWSHQDTF KCEAVNKQSF QTSQPFQVLK KKMNENLEIS ENSDDCQILT CPVCFRAQGC ISLEALN KH VDECLDGPSI SENFKMFSCS HVSATKVNKK ENVPASSLCE KQDYEAHPKI KEISSVDCIA LVDTIDNSSK AESIDALS N KHSKEECSSL PSKSFNIEHC HQNSSSTVSL ENEDVGSFRQ EYRQPYLCEV KTGQALVCPV CNVEQKTSDL TLFNVHVDV CLNKSFIQEL RKDKFNPVNQ PKESSRSTGS SSGVQKAVTR TKRPGLMTKY STSKKIKPNN PKHTLDIFFK UniProtKB: DNA polymerase kappa |
-Macromolecule #3: DNA Primer
Macromolecule | Name: DNA Primer / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 7.665987 KDa |
Sequence | String: (DA)(DG)(DC)(DT)(DA)(DT)(DG)(DA)(DC)(DC) (DA)(DT)(DG)(DA)(DT)(DT)(DA)(DC)(DG)(DA) (DA)(DT)(DT)(DG)(DOC) |
-Macromolecule #4: DNA Template
Macromolecule | Name: DNA Template / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 11.677539 KDa |
Sequence | String: (DC)(DT)(DG)(DC)(DA)(DC)(DG)(DA)(DA)(DT) (DT)(DA)(DA)(DG)(DC)(DA)(DA)(DT)(DT)(DC) (DG)(DT)(DA)(DA)(DT)(DC)(DA)(DT)(DG) (DG)(DT)(DC)(DA)(DT)(DA)(DG)(DC)(DT) |
-Macromolecule #5: THYMIDINE-5'-TRIPHOSPHATE
Macromolecule | Name: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: TTP |
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Molecular weight | Theoretical: 482.168 Da |
Chemical component information | ![]() ChemComp-TTP: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec. / Details: 40 mA | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 77.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 3889 / Average exposure time: 5.0 sec. / Average electron dose: 47.0 e/Å2 / Details: Super resolution mode & AFIS used |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000 |
Sample stage | Specimen 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
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | ![]() PDB-7nv1: |