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- EMDB-48117: Cryo-EM structure of apo-form human DNA polymerase delta -

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Entry
Database: EMDB / ID: EMD-48117
TitleCryo-EM structure of apo-form human DNA polymerase delta
Map dataSharpened map
Sample
  • Complex: Human DNA polymerase delta
    • Protein or peptide: DNA polymerase delta catalytic subunit
    • Protein or peptide: DNA polymerase delta subunit 2
    • Protein or peptide: DNA polymerase delta subunit 3
    • Protein or peptide: DNA polymerase delta subunit 4
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTER
KeywordsDNA polymerase delta / human / cryo-EM / REPLICATION
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 / 3'-5'-DNA exonuclease activity / nucleotide-excision repair, DNA gap filling / 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 / 3'-5'-DNA exonuclease activity / nucleotide-excision repair, DNA gap filling / 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 / 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) / aggresome / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / error-free translesion synthesis / DNA biosynthetic process / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / 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 / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / DNA repair / nucleotide binding / chromatin binding / enzyme binding / DNA binding / zinc ion binding / nucleoplasm / nucleus / membrane / cytosol / cytoplasm
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 polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsMurakami KS / Shin Y
Funding support United States, 2 items
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, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48117.png

Downloads & links

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Map

FileDownload / File: emd_48117.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 320 pix.
= 358.4 Å		1.12 Å/pix.
x 320 pix.
= 358.4 Å		1.12 Å/pix.
x 320 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.217
Minimum - Maximum-0.7929375 - 1.5313832
Average (Standard dev.)0.0003511145 (±0.025524538)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Non-uniform refined map without sharpening

Fileemd_48117_additional_1.map
AnnotationNon-uniform refined map without sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_48117_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_48117_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Human DNA polymerase delta

EntireName: Human DNA polymerase delta
Components
  • Complex: Human DNA polymerase delta
    • Protein or peptide: DNA polymerase delta catalytic subunit
    • Protein or peptide: DNA polymerase delta subunit 2
    • Protein or peptide: DNA polymerase delta subunit 3
    • Protein or peptide: DNA polymerase delta subunit 4
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTER

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Supramolecule #1: Human DNA polymerase delta

SupramoleculeName: Human DNA polymerase delta / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: Apo-form of the Human DNA polymerase delta
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 240.692 KDa

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Macromolecule #1: DNA polymerase delta catalytic subunit

MacromoleculeName: DNA polymerase delta catalytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 123.785922 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDGKRRPGPG PGVPPKRARG GLWDDDDAPR PSQFEEDLAL MEEMEAEHRL QEQEEEELQS VLEGVADGQV PPSAIDPRWL RPTPPALDP QTEPLIFQQL EIDHYVGPAQ PVPGGPPPSR GSVPVLRAFG VTDEGFSVCC HIHGFAPYFY TPAPPGFGPE H MGDLQREL ...String:
MDGKRRPGPG PGVPPKRARG GLWDDDDAPR PSQFEEDLAL MEEMEAEHRL QEQEEEELQS VLEGVADGQV PPSAIDPRWL RPTPPALDP QTEPLIFQQL EIDHYVGPAQ PVPGGPPPSR GSVPVLRAFG VTDEGFSVCC HIHGFAPYFY TPAPPGFGPE H MGDLQREL NLAISRDSRG GRELTGPAVL AVELCSRESM FGYHGHGPSP FLRITVALPR LVAPARRLLE QGIRVAGLGT PS FAPYEAN VDFEIRFMVD TDIVGCNWLE LPAGKYALRL KEKATQCQLE ADVLWSDVVS HPPEGPWQRI APLRVLSFDI ECA GRKGIF PEPERDPVIQ ICSLGLRWGE PEPFLRLALT LRPCAPILGA KVQSYEKEED LLQAWSTFIR IMDPDVITGY NIQN FDLPY LISRAQTLKV QTFPFLGRVA GLCSNIRDSS FQSKQTGRRD TKVVSMVGRV QMDMLQVLLR EYKLRSYTLN AVSFH FLGE QKEDVQHSII TDLQNGNDQT RRRLAVYCLK DAYLPLRLLE RLMVLVNAVE MARVTGVPLS YLLSRGQQVK VVSQLL RQA MHEGLLMPVV KSEGGEDYTG ATVIEPLKGY YDVPIATLDF SSLYPSIMMA HNLCYTTLLR PGTAQKLGLT EDQFIRT PT GDEFVKTSVR KGLLPQILEN LLSARKRAKA ELAKETDPLR RQVLDGRQLA LKVSANSVYG FTGAQVGKLP CLEISQSV T GFGRQMIEKT KQLVESKYTV ENGYSTSAKV VYGDTDSVMC RFGVSSVAEA MALGREAADW VSGHFPSPIR LEFEKVYFP YLLISKKRYA GLLFSSRPDA HDRMDCKGLE AVRRDNCPLV ANLVTASLRR LLIDRDPEGA VAHAQDVISD LLCNRIDISQ LVITKELTR AASDYAGKQA HVELAERMRK RDPGSAPSLG DRVPYVIISA AKGVAAYMKS EDPLFVLEHS LPIDTQYYLE Q QLAKPLLR IFEPILGEGR AEAVLLRGDH TRCKTVLTGK VGGLLAFAKR RNCCIGCRTV LSHQGAVCEF CQPRESELYQ KE VSHLNAL EERFSRLWTQ CQRCQGSLHE DVICTSRDCP IFYMRKKVRK DLEDQEQLLR RFGPPGPEAW

UniProtKB: DNA polymerase delta catalytic subunit

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Macromolecule #2: DNA polymerase delta subunit 2

MacromoleculeName: DNA polymerase delta subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.338168 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFSEQAAQRA HTLLSPPSAN NATFARVPVA TYTNSSQPFR LGERSFSRQY AHIYATRLIQ MRPFLENRAQ QHWGSGVGVK KLCELQPEE KCCVVGTLFK AMPLQPSILR EVSEEHNLLP QPPRSKYIHP DDELVLEDEL QRIKLKGTID VSKLVTGTVL A VFGSVRDD ...String:
MFSEQAAQRA HTLLSPPSAN NATFARVPVA TYTNSSQPFR LGERSFSRQY AHIYATRLIQ MRPFLENRAQ QHWGSGVGVK KLCELQPEE KCCVVGTLFK AMPLQPSILR EVSEEHNLLP QPPRSKYIHP DDELVLEDEL QRIKLKGTID VSKLVTGTVL A VFGSVRDD GKFLVEDYCF ADLAPQKPAP PLDTDRFVLL VSGLGLGGGG GESLLGTQLL VDVVTGQLGD EGEQCSAAHV SR VILAGNL LSHSTQSRDS INKAKYLTKK TQAASVEAVK MLDEILLQLS ASVPVDVMPG EFDPTNYTLP QQPLHPCMFP LAT AYSTLQ LVTNPYQATI DGVRFLGTSG QNVSDIFRYS SMEDHLEILE WTLRVRHISP TAPDTLGCYP FYKTDPFIFP ECPH VYFCG NTPSFGSKII RGPEDQTVLL VTVPDFSATQ TACLVNLRSL ACQPISFSGF GAEDDDLGGL GLGP

UniProtKB: DNA polymerase delta subunit 2

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Macromolecule #3: DNA polymerase delta subunit 3

MacromoleculeName: DNA polymerase delta subunit 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.486359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADQLYLENI DEFVTDQNKI VTYKWLSYTL GVHVNQAKQM LYDYVERKRK ENSGAQLHVT YLVSGSLIQN GHSCHKVAVV REDKLEAVK SKLAVTASIH VYSIQKAMLK DSGPLFNTDY DILKSNLQNC SKFSAIQCAA AVPRAPAESS SSSKKFEQSH L HMSSETQA ...String:
MADQLYLENI DEFVTDQNKI VTYKWLSYTL GVHVNQAKQM LYDYVERKRK ENSGAQLHVT YLVSGSLIQN GHSCHKVAVV REDKLEAVK SKLAVTASIH VYSIQKAMLK DSGPLFNTDY DILKSNLQNC SKFSAIQCAA AVPRAPAESS SSSKKFEQSH L HMSSETQA NNELTTNGHG PPASKQVSQQ PKGIMGMFAS KAAAKTQETN KETKTEAKEV TNASAAGNKA PGKGNMMSNF FG KAAMNKF KVNLDSEQAV KEEKIVEQPT VSVTEPKLAT PAGLKKSSKK AEPVKVLQKE KKRGKRVALS DDETKETENM RKK RRRIKL PESDSSEDEV FPDSPGAYEA ESPSPPPPPS PPLEPVPKTE PEPPSVKSSS GENKRKRKRV LKSKTYLDGE GCIV TEKVY ESESCTDSEE ELNMKTSSVH RPPAMTVKKE PREERKGPKK GTAALGKANR QVSITGFFQR K

UniProtKB: DNA polymerase delta subunit 3

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Macromolecule #4: DNA polymerase delta subunit 4

MacromoleculeName: DNA polymerase delta subunit 4 / type: protein_or_peptide / ID: 4
Details: C-terminal His6 tag was added from pET20(+) vector by inserting PolD4 gene at NdelI-XhoI digested sites
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.409202 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGRKRLITDS YPVVKRREGP AGHSKGELAP ELGEEPQPRD EEEAELELLR QFDLAWQYGP CTGITRLQRW CRAKQMGLEP PPEVWQVLK THPGDPRFQC SLWHLYPLEH HHHHH

UniProtKB: DNA polymerase delta subunit 4

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 6 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.44 mg/mL
BufferpH: 7.5 / Component - Concentration: 10.0 mM / Component - Formula: Hepes-NaOH / Component - Name: Hepes
Details: 10 mM HEPES-NaOH (pH 7.5), 120 mM NaCl, 2 % glycerol, 8 mM of CHAPSO
GridModel: Quantifoil / Material: COPPER / Mesh: 400
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Detailsglow-discharged C-Flat Holey Carbon grid (CF-2/1-4Cu-50)

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 6915 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 114792
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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