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- PDB-9f6d: Human DNA polymerase epsilon bound to DNA and PCNA (open conformation) -

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Basic information

Entry
Database: PDB / ID: 9f6d
TitleHuman DNA polymerase epsilon bound to DNA and PCNA (open conformation)
Components
  • DNA nascent strand
  • DNA polymerase epsilon catalytic subunit A
  • DNA template strand
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / DNA / polymerase / epsilon / PCNA / leading strand / human / replisome / proofreading
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 / replisome / Removal of the Flap Intermediate / MutLalpha complex binding / Telomere C-strand (Lagging Strand) Synthesis / 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 / embryonic organ development / PCNA-Dependent Long Patch Base Excision Repair / DNA repair-dependent chromatin remodeling / replication fork processing / Activation of the pre-replicative complex / response to cadmium ion / estrous cycle / base-excision repair, gap-filling / mismatch repair / cyclin-dependent protein kinase holoenzyme complex / DNA polymerase binding / epithelial cell differentiation / translesion synthesis / 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 / cellular response to hydrogen peroxide / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / heart development / cellular response to UV / 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
2',3'-dideoxyadenosine triphosphate / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA sliding clamp PCNA / DNA polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsRoske, J.J. / Yeeles, J.T.P.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis for processive daughter-strand synthesis and proofreading by the human leading-strand DNA polymerase Pol ε.
Authors: Johann J Roske / Joseph T P Yeeles /
Abstract: During chromosome replication, the nascent leading strand is synthesized by DNA polymerase epsilon (Pol ε), which associates with the sliding clamp processivity factor proliferating cell nuclear ...During chromosome replication, the nascent leading strand is synthesized by DNA polymerase epsilon (Pol ε), which associates with the sliding clamp processivity factor proliferating cell nuclear antigen (PCNA) to form a processive holoenzyme. For high-fidelity DNA synthesis, Pol ε relies on nucleotide selectivity and its proofreading ability to detect and excise a misincorporated nucleotide. Here, we present cryo-electron microscopy (cryo-EM) structures of human Pol ε in complex with PCNA, DNA and an incoming nucleotide, revealing how Pol ε associates with PCNA through its PCNA-interacting peptide box and additional unique features of its catalytic domain. Furthermore, by solving a series of cryo-EM structures of Pol ε at a mismatch-containing DNA, we elucidate how Pol ε senses and edits a misincorporated nucleotide. Our structures delineate steps along an intramolecular switching mechanism between polymerase and exonuclease activities, providing the basis for a proofreading mechanism in B-family replicative polymerases.
History
DepositionMay 1, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Dec 25, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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 nascent strand
T: DNA template strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)244,4849
Polymers243,6336
Non-polymers8513
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15680 Å2
ΔGint-96 kcal/mol
Surface area97070 Å2
MethodPISA

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Components

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Protein , 2 types, 4 molecules ABCD

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


Mass: 138137.562 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: Trichoplusia ni (cabbage looper)
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(DE3) (bacteria) / References: UniProt: P12004

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DNA chain , 2 types, 2 molecules PT

#3: DNA chain DNA nascent strand


Mass: 7074.585 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: DNA chain DNA template strand


Mass: 12033.732 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#6: Chemical ChemComp-DDS / 2',3'-dideoxyadenosine triphosphate


Mass: 475.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O11P3
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
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

Component
IDNameTypeEntity IDParent-IDSource
1Quaternary Complex of human leading strand polymerase epsilon, Proliferating cell nuclear antigen (PCNA), substrate DNA and incoming nucleotide.COMPLEX#1-#40MULTIPLE SOURCES
2DNA polymerase epsilon catalytic subunit ACOMPLEX#11RECOMBINANT
3Proliferating cell nuclear antigenCOMPLEX#21RECOMBINANT
4DNACOMPLEX#3-#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33Escherichia coli BL21(DE3) (bacteria)469008
44synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 3000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40.08 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175614 / Symmetry type: POINT
RefinementCross valid method: NONE

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