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- PDB-9f6l: Human DNA Polymerase epsilon bound to T-C mismatched DNA (Mismatc... -

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Basic information

Entry
Database: PDB / ID: 9f6l
TitleHuman DNA Polymerase epsilon bound to T-C mismatched DNA (Mismatch Excision state)
Components
  • DNA nascent strand
  • DNA polymerase epsilon catalytic subunit A
  • DNA template strand
KeywordsREPLICATION / DNA / polymerase / epsilon / PCNA / leading strand / human / replisome / proofreading
Function / homology
Function and homology information


DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA synthesis involved in DNA repair / leading strand elongation / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex ...DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA synthesis involved in DNA repair / leading strand elongation / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / embryonic organ development / base-excision repair, gap-filling / Gap-filling DNA repair synthesis and ligation in GG-NER / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / G1/S transition of mitotic cell cycle / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / DNA binding / zinc ion binding / nucleoplasm / nucleus / plasma membrane
Similarity search - Function
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 / 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 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 / 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 polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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

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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
P: DNA nascent strand
T: DNA template strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,6196
Polymers157,1873
Non-polymers4323
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 138239.609 Da / Num. of mol.: 1
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: DNA chain DNA nascent strand


Mass: 9489.113 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA template strand


Mass: 9458.104 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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-#30MULTIPLE SOURCES
2DNA polymerase epsilon catalytic subunit ACOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
43synthetic 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: 1200 nm
Image recordingElectron dose: 36.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18858 / Symmetry type: POINT
RefinementCross valid method: NONE

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