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- PDB-9ekb: Cryo-EM structure of apo-form human DNA polymerase delta -

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Basic information

Entry
Database: PDB / ID: 9ekb
TitleCryo-EM structure of apo-form human DNA polymerase delta
Components
  • (DNA polymerase delta subunit ...) x 3
  • DNA polymerase delta catalytic subunit
KeywordsREPLICATION / DNA polymerase delta / human / cryo-EM
Function / homology
Function and homology information


delta DNA polymerase complex / DNA synthesis involved in UV-damage excision repair / zeta DNA polymerase complex / nucleotide-excision repair complex / Cytosolic iron-sulfur cluster assembly / nucleotide-excision repair, DNA gap filling / 3'-5'-DNA exonuclease activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand ...delta DNA polymerase complex / DNA synthesis involved in UV-damage excision repair / zeta DNA polymerase complex / nucleotide-excision repair complex / Cytosolic iron-sulfur cluster assembly / nucleotide-excision repair, DNA gap filling / 3'-5'-DNA exonuclease activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / DNA replication proofreading / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Removal of the Flap Intermediate from the C-strand / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / aggresome / DNA biosynthetic process / error-free translesion synthesis / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / PCNA-Dependent Long Patch Base Excision Repair / fatty acid homeostasis / error-prone translesion synthesis / mismatch repair / response to UV / base-excision repair, gap-filling / positive regulation of endothelial cell proliferation / Gap-filling DNA repair synthesis and ligation in GG-NER / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / 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 / cellular response to UV / 4 iron, 4 sulfur cluster binding / protein-macromolecule adaptor activity / DNA-directed DNA polymerase / damaged DNA binding / DNA-directed DNA polymerase activity / DNA replication / DNA repair / nucleotide binding / chromatin binding / enzyme binding / DNA binding / zinc ion binding / nucleoplasm / nucleus / membrane / cytoplasm / cytosol
Similarity search - Function
DNA polymerase delta, subunit 4 / DNA polymerase delta, subunit 4 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 3 superfamily / DNA polymerase subunit Cdc27 / DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta ...DNA polymerase delta, subunit 4 / DNA polymerase delta, subunit 4 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 3 superfamily / DNA polymerase subunit Cdc27 / DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / : / C4-type zinc-finger of DNA polymerase delta / DNA polymerase delta catalytic subunit-like, N-terminal domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / : / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / DNA polymerase family B / 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 polymerase delta catalytic subunit / DNA polymerase delta subunit 2 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsMurakami, K.S. / Shin, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131860 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM147238 United States
CitationJournal: J Biol Chem / Year: 2025
Title: Cryo-EM structure of apo-form human DNA polymerase δ elucidates its minimal DNA synthesis activity without PCNA.
Authors: Yeonoh Shin / Mark Hedglin / Katsuhiko S Murakami /
Abstract: DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication and genome maintenance, essential for lagging strand synthesis, leading strand initiation, and DNA repair. While human Pol δ ...DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication and genome maintenance, essential for lagging strand synthesis, leading strand initiation, and DNA repair. While human Pol δ exhibits high activity and processivity in its holoenzyme form complexed with proliferating cell nuclear antigen (PCNA), it shows minimal DNA synthesis activity without PCNA, the molecular basis of which remains unclear. Here, we present the cryo-EM structure of the apo-form human Pol δ, comprising the catalytic subunit p125 and regulatory subunits p66, p50, and p12, at an overall resolution of 3.65 Å. We identified an acidic α-helix at the N terminus of p125, which occupies the single-stranded DNA-binding cavity within the polymerase domain in the apo-form Pol δ. This interaction likely inhibits DNA binding in the absence of PCNA, explaining the low activity of apo-form Pol δ. The acidic α-helix is absent in yeast Pol δ, providing a molecular explanation for species-specific differences in PCNA-independent Pol δ activity. These findings provide critical insights into the regulatory mechanisms of Pol δ and its reliance on PCNA for efficient DNA synthesis.
History
DepositionDec 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Apr 2, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _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 delta catalytic subunit
B: DNA polymerase delta subunit 2
C: DNA polymerase delta subunit 3
D: DNA polymerase delta subunit 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,4376
Polymers240,0204
Non-polymers4172
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

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Protein , 1 types, 1 molecules A

#1: Protein DNA polymerase delta catalytic subunit / 3'-5' exodeoxyribonuclease / DNA polymerase subunit delta p125


Mass: 123785.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD1, POLD / Plasmid: pET-POLD4/1 / Details (production host): Amp resistant / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-codonplus (DE3)-RP
References: UniProt: P28340, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters

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DNA polymerase delta subunit ... , 3 types, 3 molecules BCD

#2: Protein DNA polymerase delta subunit 2 / DNA polymerase delta subunit p50


Mass: 51338.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD2 / Plasmid: pGBM-POLD2/3 / Details (production host): streptomycin resistant / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-codonplus (DE3)-RP / References: UniProt: P49005
#3: Protein DNA polymerase delta subunit 3 / DNA polymerase delta subunit C / DNA polymerase delta subunit p66 / DNA polymerase delta subunit p68


Mass: 51486.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD3, KIAA0039 / Plasmid: pGBM-POLD2/3 / Details (production host): streptomycin resistant / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-codonplus (DE3)-RP / References: UniProt: Q15054
#4: Protein DNA polymerase delta subunit 4 / DNA polymerase delta subunit p12


Mass: 13409.202 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal His6 tag was added from pET20(+) vector by inserting PolD4 gene at NdelI-XhoI digested sites
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD4, POLDS / Plasmid: pET-POLD4/1 / Details (production host): Amp resistant / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-codonplus (DE3)-RP / References: UniProt: Q9HCU8

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: 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

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Details

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: Human DNA polymerase delta / Type: COMPLEX / Details: Apo-form of the Human DNA polymerase delta / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.240692 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-codonplus (DE3)-RP / Plasmid: pET-POLD4/1 and pGBM-POLD2/3
Buffer solutionpH: 7.5
Details: 10 mM HEPES-NaOH (pH 7.5), 120 mM NaCl, 2 % glycerol, 8 mM of CHAPSO
Buffer componentConc.: 10 mM / Name: Hepes / Formula: Hepes-NaOH
SpecimenConc.: 1.44 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: glow-discharged C-Flat Holey Carbon grid (CF-2/1-4Cu-50)
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: OTHER
Electron lensMode: OTHER / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6915

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Processing

EM softwareName: PHENIX / Version: dev_5330 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114792 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE

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