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- PDB-9b8s: Human polymerase epsilon bound to PCNA and DNA in the nucleotide ... -

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Basic information

Entry
Database: PDB / ID: 9b8s
TitleHuman polymerase epsilon bound to PCNA and DNA in the nucleotide exchange state
Components
  • DNA (5'-D(P*AP*AP*AP*GP*TP*GP*AP*AP*AP*AP*AP*TP*CP*TP*AP*AP*AP*GP*CP*AP*TP*CP*AP*C)-3')
  • DNA (5'-D(P*GP*TP*GP*AP*TP*GP*CP*TP*TP*TP*AP*GP*AP*TP*TP*TP*TP*TP*C)-3')
  • DNA polymerase epsilon catalytic subunit A
  • Proliferating cell nuclear antigen
KeywordsDNA Binding Protein/DNA / DNA polymerase / DNA / DNA Binding Protein-DNA complex
Function / homology
Function and homology information


DNA replication initiation / epsilon DNA polymerase complex / 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 / nucleotide-excision repair, DNA gap filling / nuclear lamina / positive regulation of DNA-directed DNA polymerase activity ...DNA replication initiation / epsilon DNA polymerase complex / 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 / nucleotide-excision repair, DNA gap filling / nuclear lamina / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / MutLalpha complex binding / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / DNA replication proofreading / PCNA complex / single-stranded DNA 3'-5' DNA exonuclease activity / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / 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 / replisome / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / response to L-glutamate / DNA synthesis involved in DNA repair / histone acetyltransferase binding / DNA polymerase processivity factor activity / leading strand elongation / G1/S-Specific Transcription / response to dexamethasone / replication fork processing / nuclear replication fork / SUMOylation of DNA replication proteins / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / embryonic organ development / translesion synthesis / mismatch repair / response to cadmium ion / estrous cycle / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / epithelial cell differentiation / positive regulation of DNA repair / male germ cell nucleus / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / liver regeneration / replication fork / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / cellular response to hydrogen peroxide / DNA-templated DNA replication / G1/S transition of mitotic cell cycle / 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 / mitotic cell cycle / heart development / 4 iron, 4 sulfur cluster binding / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / nuclear body / nucleotide binding / centrosome / chromatin binding / protein-containing complex binding / chromatin / enzyme binding / negative regulation of transcription by RNA polymerase II / DNA binding / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / nucleus / plasma membrane
Similarity search - Function
Zinc finger domain of DNA polymerase-epsilon / : / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / Proliferating cell nuclear antigen signature 2. ...Zinc finger domain of DNA polymerase-epsilon / : / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / Proliferating cell nuclear antigen signature 2. / 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 / DNA polymerase family B, thumb domain / : / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / Proliferating cell nuclear antigen / DNA polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesHomo sapiens (human)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.01 Å
AuthorsWang, F. / He, Q. / Li, H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM148159 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structures of the human leading strand Polε-PCNA holoenzyme.
Authors: Qing He / Feng Wang / Nina Y Yao / Michael E O'Donnell / Huilin Li /
Abstract: In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the ...In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Polε catalytic domain has been determined, how Polε interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Polε-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Polε catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Polε. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding.
History
DepositionMar 31, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.3Oct 2, 2024Group: Data collection / Database references / Category: citation_author / em_admin
Item: _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase epsilon catalytic subunit A
B: Proliferating cell nuclear antigen
C: Proliferating cell nuclear antigen
D: Proliferating cell nuclear antigen
P: DNA (5'-D(P*GP*TP*GP*AP*TP*GP*CP*TP*TP*TP*AP*GP*AP*TP*TP*TP*TP*TP*C)-3')
T: DNA (5'-D(P*AP*AP*AP*GP*TP*GP*AP*AP*AP*AP*AP*TP*CP*TP*AP*AP*AP*GP*CP*AP*TP*CP*AP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)377,4537
Polymers377,1016
Non-polymers3521
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein DNA polymerase epsilon catalytic subunit A / 3'-5' exodeoxyribonuclease / DNA polymerase II subunit A


Mass: 261753.234 Da / Num. of mol.: 1 / Mutation: D275A, E277A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLE, POLE1
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q07864, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters
#2: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


Mass: 28795.752 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PCNA
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P12004
#3: DNA chain DNA (5'-D(P*GP*TP*GP*AP*TP*GP*CP*TP*TP*TP*AP*GP*AP*TP*TP*TP*TP*TP*C)-3')


Mass: 10823.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) DNA molecule (others) / Production host: DNA molecule (others)
#4: DNA chain DNA (5'-D(P*AP*AP*AP*GP*TP*GP*AP*AP*AP*AP*AP*TP*CP*TP*AP*AP*AP*GP*CP*AP*TP*CP*AP*C)-3')


Mass: 18136.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) DNA molecule (others) / Production host: DNA molecule (others)
#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: The DNA bound Pol epsilon and PCNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.45 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2100 mMSodium chlorideNaCl1
31 mMDithiothreitolDTT1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280 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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4.4.0particle selectionBlob picker and Template picker were used to select particles
4cryoSPARCv4.4.0CTF correction
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71210 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416788
ELECTRON MICROSCOPYf_angle_d0.922898
ELECTRON MICROSCOPYf_dihedral_angle_d15.8922651
ELECTRON MICROSCOPYf_chiral_restr0.0552553
ELECTRON MICROSCOPYf_plane_restr0.0072779

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