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- PDB-9ne6: Human polymerase epsilon bound to PCNA and DNA with an in-situ-ge... -

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Basic information

Entry
Database: PDB / ID: 9ne6
TitleHuman polymerase epsilon bound to PCNA and DNA with an in-situ-generated mismatch in the mismatch-editing state
Components
  • DNA (33-MER)
  • DNA (47-MER)
  • DNA polymerase epsilon catalytic subunit A
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / DNA polymerase / exo / holoenzyme / DNA
Function / homology
Function and homology information


DNA replication initiation / epsilon DNA polymerase complex / 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 / Polymerase switching / Processive synthesis on the lagging strand ...DNA replication initiation / epsilon DNA polymerase complex / 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 / Polymerase switching / Processive synthesis on the lagging strand / DNA replication proofreading / PCNA complex / single-stranded DNA 3'-5' DNA exonuclease activity / MutLalpha complex binding / Removal of the Flap Intermediate / Telomere C-strand (Lagging Strand) Synthesis / replisome / 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 / mitotic DNA replication / response to L-glutamate / Processive synthesis on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / response to dexamethasone / histone acetyltransferase binding / DNA synthesis involved in DNA repair / leading strand elongation / DNA polymerase processivity factor activity / G1/S-Specific Transcription / nuclear replication fork / SUMOylation of DNA replication proteins / replication fork processing / DNA repair-dependent chromatin remodeling / PCNA-Dependent Long Patch Base Excision Repair / embryonic organ development / Activation of the pre-replicative complex / response to cadmium ion / estrous cycle / mismatch repair / base-excision repair, gap-filling / cyclin-dependent protein kinase holoenzyme complex / DNA polymerase binding / translesion synthesis / epithelial cell differentiation / liver regeneration / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / positive regulation of DNA replication / nuclear estrogen receptor binding / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / replication fork / G1/S transition of mitotic cell cycle / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / receptor tyrosine kinase binding / Recognition of DNA damage by PCNA-containing replication complex / DNA-templated DNA replication / HDR through Homologous Recombination (HRR) / cellular response to hydrogen peroxide / Dual Incision in GG-NER / cellular response to UV / heart development / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / response to estradiol / mitotic cell cycle / E3 ubiquitin ligases ubiquitinate target proteins / 4 iron, 4 sulfur cluster binding / chromatin organization / DNA-directed DNA polymerase / damaged DNA binding / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / nucleotide binding / centrosome / chromatin binding / chromatin / protein-containing complex binding / enzyme binding / DNA binding / extracellular exosome / nucleoplasm / zinc ion binding / identical protein binding / nucleus / plasma membrane
Similarity search - Function
: / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / 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. ...: / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / 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, C-terminal / Proliferating cell nuclear antigen, C-terminal domain / 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, N-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) / DNA sliding clamp PCNA / DNA polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 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: Proc Natl Acad Sci U S A / Year: 2025
Title: The proofreading mechanism of the human leading-strand DNA polymerase ε holoenzyme.
Authors: Feng Wang / Qing He / Michael E O'Donnell / Huilin Li /
Abstract: The eukaryotic leading-strand DNA polymerase ε (Polε) is a dual-function enzyme with a proofreading 3'-5' exonuclease () site located 40 Å from the DNA synthesizing site. Errors in Polε ...The eukaryotic leading-strand DNA polymerase ε (Polε) is a dual-function enzyme with a proofreading 3'-5' exonuclease () site located 40 Å from the DNA synthesizing site. Errors in Polε proofreading can cause various mutations, including C-to-G transversions, the most prevalent mutation in cancers and genetic diseases. Polε interacts with all three subunits of the PCNA ring to assemble a functional holoenzyme. Despite previous studies on proofreading of several Pol's, how Polε-or any Pol complexed with its sliding clamp-proofreads a mismatch generated in situ has been unknown. We show here by cryo-EM that a template/primer DNA substrate with a preexisting mismatch cannot enter the site of Polε-PCNA holoenzyme, but a mismatch generated in situ in the site yields three bona fide proofreading intermediates of Polε-PCNA holoenzyme. These intermediates reveal how the mismatch is dislodged from the site, how the DNA unwinds six base pairs, and how the unpaired primer 3'-end is inserted into the site for cleavage. These results unexpectedly demonstrate that PCNA imposes strong steric constraints that extend unwinding and direct the trajectory of mismatched DNA and that this trajectory is dramatically different than for Polε in the absence of PCNA. These findings suggest a physiologically relevant proofreading mechanism for the human Polε holoenzyme.
History
DepositionFeb 19, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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 (33-MER)
T: DNA (47-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)249,5327
Polymers249,1816
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: 138137.562 Da / Num. of mol.: 1
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: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P12004
#3: DNA chain DNA (33-MER)


Mass: 10236.587 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (47-MER)


Mass: 14419.317 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (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
Has protein modificationN

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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: Ternary complex of human DNA polymerase epsilon with PCNA and the mismatched DNA
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
225 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 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 softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 15815155
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 296448 / Symmetry type: POINT
RefinementHighest resolution: 3.11 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416772
ELECTRON MICROSCOPYf_angle_d0.86622879
ELECTRON MICROSCOPYf_dihedral_angle_d17.4042648
ELECTRON MICROSCOPYf_chiral_restr0.0532551
ELECTRON MICROSCOPYf_plane_restr0.0052772

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