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- EMDB-49300: Human polymerase epsilon bound to PCNA and DNA with an in-situ-ge... -

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Basic information

Entry
Database: EMDB / ID: EMD-49300
TitleHuman polymerase epsilon bound to PCNA and DNA with an in-situ-generated mismatch in the Pol-backtracking state
Map dataFinal sharpened EM map by cryosparc
Sample
  • Complex: Ternary complex of human DNA polymerase epsilon with PCNA and the mismatched DNA
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: DNA (33-MER)
    • DNA: DNA (47-MER)
  • Ligand: IRON/SULFUR CLUSTER
KeywordsDNA polymerase / exo / holoenzyme / DNA / REPLICATION
Function / homology
Function and homology information


DNA replication initiation / epsilon DNA polymerase complex / positive regulation of deoxyribonuclease activity / 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 / positive regulation of DNA-directed DNA polymerase activity ...DNA replication initiation / epsilon DNA polymerase complex / positive regulation of deoxyribonuclease activity / 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 / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / MutLalpha complex binding / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / DNA replication proofreading / PCNA complex / single-stranded DNA 3'-5' DNA exonuclease activity / 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 / Transcription of E2F targets under negative control by DREAM complex / replisome / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / response to L-glutamate / histone acetyltransferase binding / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / leading strand elongation / G1/S-Specific Transcription / response to dexamethasone / replication fork processing / nuclear replication fork / SUMOylation of DNA replication proteins / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / embryonic organ development / mismatch repair / translesion synthesis / response to cadmium ion / estrous cycle / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / epithelial cell differentiation / positive regulation of DNA repair / male germ cell nucleus / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / liver regeneration / replication fork / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / cellular response to hydrogen peroxide / DNA-templated DNA replication / G1/S transition of mitotic cell cycle / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / mitotic cell cycle / heart development / 4 iron, 4 sulfur cluster binding / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / nuclear body / nucleotide binding / centrosome / chromatin binding / protein-containing complex binding / chromatin / enzyme binding / negative regulation of transcription by RNA polymerase II / DNA binding / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / nucleus / plasma membrane
Similarity search - Function
Zinc finger domain of DNA polymerase-epsilon / : / : / 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 / Proliferating cell nuclear antigen signature 2. ...Zinc finger domain of DNA polymerase-epsilon / : / : / 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 / Proliferating cell nuclear antigen signature 2. / 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, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-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
Proliferating cell nuclear antigen / DNA polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsWang F / He Q / Li H
Funding support United States, 2 items
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, 2025-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49300.png

Downloads & links

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Map

FileDownload / File: emd_49300.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal sharpened EM map by cryosparc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 264.96 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.095
Minimum - Maximum-0.6383154 - 1.212012
Average (Standard dev.)0.00069511787 (±0.02284055)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened EM map

Fileemd_49300_additional_1.map
AnnotationUnsharpened EM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_49300_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_49300_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of human DNA polymerase epsilon with PCNA and the...

EntireName: Ternary complex of human DNA polymerase epsilon with PCNA and the mismatched DNA
Components
  • Complex: Ternary complex of human DNA polymerase epsilon with PCNA and the mismatched DNA
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: DNA (33-MER)
    • DNA: DNA (47-MER)
  • Ligand: IRON/SULFUR CLUSTER

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Supramolecule #1: Ternary complex of human DNA polymerase epsilon with PCNA and the...

SupramoleculeName: Ternary complex of human DNA polymerase epsilon with PCNA and the mismatched DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: DNA polymerase epsilon catalytic subunit A

MacromoleculeName: DNA polymerase epsilon catalytic subunit A / 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: 138.137562 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MSLRSGGRRR ADPGADGEAS RDDGATSSVS ALKRLERSQW TDKMDLRFGF ERLKEPGEKT GWLINMHPTE ILDEDKRLGS AVDYYFIQD DGSRFKVALP YKPYFYIATR KGCEREVSSF LSKKFQGKIA KVETVPKEDL DLPNHLVGLK RNYIRLSFHT V EDLVKVRK ...String:
MSLRSGGRRR ADPGADGEAS RDDGATSSVS ALKRLERSQW TDKMDLRFGF ERLKEPGEKT GWLINMHPTE ILDEDKRLGS AVDYYFIQD DGSRFKVALP YKPYFYIATR KGCEREVSSF LSKKFQGKIA KVETVPKEDL DLPNHLVGLK RNYIRLSFHT V EDLVKVRK EISPAVKKNR EQDHASDAYT ALLSSVLQRG GVITDEEETS KKIADQLDNI VDMREYDVPY HIRLSIDLKI HV AHWYNVR YRGNAFPVEI TRRDDLVERP DPVVLAFAIA TTKLPLKFPD AETDQIMMIS YMIDGQGYLI TNREIVSEDI EDF EFTPKP EYEGPFCVFN EPDEAHLIQR WFEHVQETKP TIMVTYNGDF FDWPFVEARA AVHGLSMQQE IGFQKDSQGE YKAP QCIHM DCLRWVKRDS YLPVGSHNLK AAAKAKLGYD PVELDPEDMC RMATEQPQTL ATYSVSDAVA TYYLYMKYVH PFIFA LCTI IPMEPDEVLR KGSGTLCEAL LMVQAFHANI IFPNKQEQEF NKLTDDGHVL DSETYVGGHV EALESGVFRS DIPCRF RMN PAAFDFLLQR VEKTLRHALE EEEKVPVEQV TNFEEVCDEI KSKLASLKDV PSRIECPLIY HLDVGAMYPN IILTNRL QP SAMVDEATCA ACDFNKPGAN CQRKMAWQWR GEFMPASRSE YHRIQHQLES EKFPPLFPEG PARAFHELSR EEQAKYEK R RLADYCRKAY KKIHITKVEE RLTTICQREN SFYVDTVRAF RDRRYEFKGL HKVWKKKLSA AVEVGDAAEV KRCKNMEVL YDSLQLAHKC ILNSFYGYVM RKGARWYSME MAGIVCFTGA NIITQARELI EQIGRPLELD TDGIWCVLPN SFPENFVFKT TNVKKPKVT ISYPGAMLNI MVKEGFTNDQ YQELAEPSSL TYVTRSENSI FFEVDGPYLA MILPASKEEG KKLKKRYAVF N EDGSLAEL KGFEVKRRGE LQLIKIFQSS VFEAFLKGST LEEVYGSVAK VADYWLDVLY SKAANMPDSE LFELISENRS MS RKLEDYG EQKSTSISTA KRLAEFLGDQ MVKDAGLSCR YIISRKPEGS PVTERAIPLA IFQAEPTVRK HFLRKWLKSS SLQ DFDIRA ILDWDYYIER LGSAIQKIIT IPAALQQVKN PVPRVKHPDW LHKKLLEKND VYKQKKISEL FTLEGRRQVT MAEA

UniProtKB: DNA polymerase epsilon catalytic subunit A

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Macromolecule #2: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.795752 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MFEARLVQGS ILKKVLEALK DLINEACWDI SSSGVNLQSM DSSHVSLVQL TLRSEGFDTY RCDRNLAMGV NLTSMSKILK CAGNEDIIT LRAEDNADTL ALVFEAPNQE KVSDYEMKLM DLDVEQLGIP EQEYSCVVKM PSGEFARICR DLSHIGDAVV I SCAKDGVK ...String:
MFEARLVQGS ILKKVLEALK DLINEACWDI SSSGVNLQSM DSSHVSLVQL TLRSEGFDTY RCDRNLAMGV NLTSMSKILK CAGNEDIIT LRAEDNADTL ALVFEAPNQE KVSDYEMKLM DLDVEQLGIP EQEYSCVVKM PSGEFARICR DLSHIGDAVV I SCAKDGVK FSASGELGNG NIKLSQTSNV DKEEEAVTIE MNEPVQLTFA LRYLNFFTKA TPLSSTVTLS MSADVPLVVE YK IADMGHL KYYLAPKIED EEGS

UniProtKB: Proliferating cell nuclear antigen

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Macromolecule #3: DNA (33-MER)

MacromoleculeName: DNA (33-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.236587 KDa
SequenceString:
(DT)(DG)(DA)(DG)(DG)(DT)(DT)(DC)(DA)(DG) (DC)(DA)(DA)(DG)(DG)(DT)(DG)(DA)(DT)(DG) (DC)(DT)(DT)(DT)(DA)(DG)(DA)(DT)(DT) (DT)(DT)(DT)(DT)

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Macromolecule #4: DNA (47-MER)

MacromoleculeName: DNA (47-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.419317 KDa
SequenceString:
(DG)(DC)(DC)(DA)(DG)(DC)(DA)(DG)(DC)(DA) (DA)(DA)(DG)(DT)(DG)(DA)(DA)(DA)(DA)(DA) (DT)(DC)(DT)(DA)(DA)(DA)(DG)(DC)(DA) (DT)(DC)(DA)(DC)(DC)(DT)(DT)(DG)(DC)(DT) (DG) (DA)(DA)(DC)(DC)(DT)(DC)(DA)

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / 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

BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15815155
CTF correctionType: NONE
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

9b8s

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 132717
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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