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- EMDB-6339: Molecular architecture of the Ub-PCNA/Pol eta complex bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-6339
TitleMolecular architecture of the Ub-PCNA/Pol eta complex bound to DNA
Map dataReconstruction of Ub-PCNA/Pol eta/DNA ternary complex
Sample
  • Sample: Ub-PCNA/Pol eta/DNA
  • Protein or peptide: Monoubiquitinated PCNA
  • Protein or peptide: Pol eta
KeywordsTranslesion synthesis / DNA damage tolerance / Y-Family polymerase / PCNA monoubiquitination
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 / MutLalpha complex binding / nuclear lamina / 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 / positive regulation of DNA-directed DNA polymerase activity / MutLalpha complex binding / nuclear lamina / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / PCNA complex / Processive synthesis on the C-strand of the telomere / Removal of the Flap Intermediate / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Polymerase switching on the C-strand of the telomere / Transcription of E2F targets under negative control by DREAM complex / Removal of the Flap Intermediate from the C-strand / replisome / response to UV-C / response to L-glutamate / histone acetyltransferase binding / leading strand elongation / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / error-free translesion synthesis / replication fork processing / G1/S-Specific Transcription / response to dexamethasone / nuclear replication fork / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / mismatch repair / cyclin-dependent protein kinase holoenzyme complex / cellular response to UV-C / pyrimidine dimer repair / translesion synthesis / error-prone translesion synthesis / positive regulation of DNA replication / regulation of DNA repair / response to cadmium ion / DNA polymerase binding / base-excision repair, gap-filling / positive regulation of DNA repair / epithelial cell differentiation / Translesion synthesis by REV1 / Translesion synthesis by POLK / 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 / 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 / response to radiation / 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 / site of double-strand break / heart development / DNA replication / chromosome, telomeric region / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nuclear body / DNA repair / centrosome / 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 / cytosol
Similarity search - Function
Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site / Ubiquitin-Binding Zinc Finger / 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 / DNA polymerase eta, ubiquitin-binding zinc finger ...Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site / Ubiquitin-Binding Zinc Finger / 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 / DNA polymerase eta, ubiquitin-binding zinc finger / Zinc finger UBZ3-type profile. / DNApol eta/Rev1, HhH motif / : / 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 eta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsLau WCY / Li Y / Zhang Q / Huen MSY
CitationJournal: Sci Rep / Year: 2015
Title: Molecular architecture of the Ub-PCNA/Pol η complex bound to DNA.
Authors: Wilson C Y Lau / Yinyin Li / Qinfen Zhang / Michael S Y Huen /
Abstract: Translesion synthesis (TLS) is the mechanism by which DNA polymerases replicate through unrepaired DNA lesions. TLS is activated by monoubiquitination of the homotrimeric proliferating cell nuclear ...Translesion synthesis (TLS) is the mechanism by which DNA polymerases replicate through unrepaired DNA lesions. TLS is activated by monoubiquitination of the homotrimeric proliferating cell nuclear antigen (PCNA) at lysine-164, followed by the switch from replicative to specialized polymerases at DNA damage sites. Pol η belongs to the Y-Family of specialized polymerases that can efficiently bypass UV-induced lesions. Like other members of the Y-Family polymerases, its recruitment to the damaged sites is mediated by the interaction with monoubiquitinated PCNA (Ub-PCNA) via its ubiquitin-binding domain and non-canonical PCNA-interacting motif in the C-terminal region. The structural determinants underlying the direct recognition of Ub-PCNA by Pol η, or Y-Family polymerases in general, remain largely unknown. Here we report a structure of the Ub-PCNA/Pol η complex bound to DNA determined by single-particle electron microscopy (EM). The overall obtained structure resembles that of the editing PCNA/PolB complex. Analysis of the map revealed the conformation of ubiquitin that binds the C-terminal domain of Pol η. Our present study suggests that the Ub-PCNA/Pol η interaction requires the formation of a structured binding interface, which is dictated by the inherent flexibility of Ub-PCNA.
History
DepositionMay 18, 2015-
Header (metadata) releaseAug 26, 2015-
Map releaseNov 18, 2015-
UpdateDec 9, 2015-
Current statusDec 9, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.27
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.27
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3ja9, PDB-3jaa
  • Surface level: 1.27
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3ja9
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jaa
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6339.gif

Downloads & links

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Map

FileDownload / File: emd_6339.map.gz / Format: CCP4 / Size: 4.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Ub-PCNA/Pol eta/DNA ternary complex
Voxel sizeX=Y=Z: 2.14 Å
Density
Contour LevelBy AUTHOR: 1.27 / Movie #1: 1.27
Minimum - Maximum-0.31146783 - 2.77413011
Average (Standard dev.)0.0 (±0.31292593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-52-52-52
Dimensions104104104
Spacing104104104
CellA=B=C: 222.56001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.142.142.14
M x/y/z104104104
origin x/y/z0.0000.0000.000
length x/y/z222.560222.560222.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-8211244
NX/NY/NZ115138149
MAP C/R/S123
start NC/NR/NS-52-52-52
NC/NR/NS104104104
D min/max/mean-0.3112.7740.000

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Supplemental data

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Sample components

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Entire : Ub-PCNA/Pol eta/DNA

EntireName: Ub-PCNA/Pol eta/DNA
Components
  • Sample: Ub-PCNA/Pol eta/DNA
  • Protein or peptide: Monoubiquitinated PCNA
  • Protein or peptide: Pol eta

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Supramolecule #1000: Ub-PCNA/Pol eta/DNA

SupramoleculeName: Ub-PCNA/Pol eta/DNA / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: Monomeric catalytic core of Pol eta binds to one homotrimeric Ub-PCNA
Number unique components: 2
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa / Method: Chemical crosslinking, SDS-PAGE

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Macromolecule #1: Monoubiquitinated PCNA

MacromoleculeName: Monoubiquitinated PCNA / type: protein_or_peptide / ID: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 120 KDa / Theoretical: 120 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)

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Macromolecule #2: Pol eta

MacromoleculeName: Pol eta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: RW644 / Recombinant plasmid: pJM879

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8 / Details: 50mM Tris-HCl, 5mM MgCl2
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on two 20ul drops of 2% w/v uranyl acetate solution for 30 seconds
GridDetails: Continuous carbon coated copper grids (Ted Pella), glow-discharged for 30 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2010
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateNov 12, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 18 e/Å2

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Image processing

CTF correctionDetails: Particles
Final two d classificationNumber classes: 227
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 7330

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Atomic model buiding 1

Initial modelPDB ID:

1vym

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3ja9:
Structure of native human PCNA

PDB-3jaa:
HUMAN DNA POLYMERASE ETA in COMPLEX WITH NORMAL DNA AND INCO NUCLEOTIDE (NRM)

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Atomic model buiding 2

Initial modelPDB ID:

3mr2

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3ja9:
Structure of native human PCNA

PDB-3jaa:
HUMAN DNA POLYMERASE ETA in COMPLEX WITH NORMAL DNA AND INCO NUCLEOTIDE (NRM)

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