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- EMDB-12602: Human Pol Kappa holoenzyme with Ub-PCNA -

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Basic information

Entry
Database: EMDB / ID: EMD-12602
TitleHuman Pol Kappa holoenzyme with Ub-PCNA
Map data
Sample
  • Complex: Pol kappa holoenzyme with Ub-PCNA
    • Complex: Pol kappa holoenzyme
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: DNA polymerase kappaPOLK
    • Complex: DNA
      • DNA: DNA PrimerPrimer (molecular biology)
      • DNA: DNA Template
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
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
Proliferating cell nuclear antigen / DNA polymerase kappa
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.4 Å
AuthorsLancey C / De Biasio A / Hamdan SM
Funding support United Kingdom, 1 items
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, 2021-
Header (metadata) releaseNov 3, 2021-
Map releaseNov 3, 2021-
UpdateNov 3, 2021-
Current statusNov 3, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nv1
  • Surface level: 2.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12602.png

Downloads & links

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Map

FileDownload / File: emd_12602.map.gz / Format: CCP4 / Size: 9.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.086 Å
Density
Contour LevelBy AUTHOR: 2.1 / Movie #1: 2.1
Minimum - Maximum-11.104069 - 15.502563
Average (Standard dev.)1.4998444e-13 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin919679
Dimensions138117149
Spacing149138117
CellA: 161.814 Å / B: 149.868 Å / C: 127.062 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0861.0861.086
M x/y/z149138117
origin x/y/z0.0000.0000.000
length x/y/z161.814149.868127.062
α/β/γ90.00090.00090.000
start NX/NY/NZ799196
NX/NY/NZ149138117
MAP C/R/S321
start NC/NR/NS969179
NC/NR/NS117138149
D min/max/mean-11.10415.5030.000

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Supplemental data

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Sample components

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Entire : Pol kappa holoenzyme with Ub-PCNA

EntireName: Pol kappa holoenzyme with Ub-PCNA
Components
  • Complex: Pol kappa holoenzyme with Ub-PCNA
    • Complex: Pol kappa holoenzyme
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: DNA polymerase kappaPOLK
    • Complex: DNA
      • DNA: DNA PrimerPrimer (molecular biology)
      • DNA: DNA Template
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: Pol kappa holoenzyme with Ub-PCNA

SupramoleculeName: Pol kappa holoenzyme with Ub-PCNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightTheoretical: 232 KDa

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Supramolecule #2: Pol kappa holoenzyme

SupramoleculeName: Pol kappa holoenzyme / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 1 / 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

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Macromolecule #2: DNA polymerase kappa

MacromoleculeName: DNA polymerase kappa / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 98.952695 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #3: DNA Primer

MacromoleculeName: DNA Primer / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.665987 KDa
SequenceString:
(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)

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Macromolecule #4: DNA Template

MacromoleculeName: DNA Template / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.677539 KDa
SequenceString:
(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)

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Macromolecule #5: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate

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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 mMC8H18N2O4SHEPES
100.0 mMCH3COOKPotassium acetate
10.0 mMCaCl2Calcium chloride
0.02 %H(C2H4O)nO(C6H4)C9H19NP-40
0.5 mMC9H15O6Ptris(2-carboxyethyl)phosphine
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 40 mA
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: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
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: 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5275717
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER / Details: Relion initial 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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 25234
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7nv1:
Human Pol Kappa holoenzyme with Ub-PCNA

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