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- EMDB-50222: Human DNA polymerase epsilon bound to DNA and PCNA (open conformation) -

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Entry
Database: EMDB / ID: EMD-50222
TitleHuman DNA polymerase epsilon bound to DNA and PCNA (open conformation)
Map data
Sample
  • Complex: Quaternary Complex of human leading strand polymerase epsilon, Proliferating cell nuclear antigen (PCNA), substrate DNA and incoming nucleotide.
    • Complex: DNA polymerase epsilon catalytic subunit A
      • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Complex: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
    • Complex: DNA
      • DNA: DNA nascent strand
      • DNA: DNA template strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: 2',3'-dideoxyadenosine triphosphate
  • Ligand: MAGNESIUM ION
KeywordsDNA / polymerase / epsilon / PCNA / leading strand / human / replication / replisome / proofreading
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 / Processive synthesis on the lagging strand / DNA replication proofreading / Telomere C-strand (Lagging Strand) Synthesis / PCNA complex / single-stranded DNA 3'-5' DNA exonuclease activity / Removal of the Flap Intermediate / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Processive synthesis on the C-strand of the telomere / Transcription of E2F targets under negative control by DREAM complex / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / replisome / response to L-glutamate / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / response to dexamethasone / DNA synthesis involved in DNA repair / histone acetyltransferase binding / DNA polymerase processivity factor activity / G1/S-Specific Transcription / leading strand elongation / nuclear replication fork / replication fork processing / SUMOylation of DNA replication proteins / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / embryonic organ development / response to cadmium ion / translesion synthesis / estrous cycle / mismatch repair / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / liver regeneration / epithelial cell differentiation / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / positive regulation of DNA replication / Gap-filling DNA repair synthesis and ligation in GG-NER / replication fork / nuclear estrogen receptor binding / male germ cell nucleus / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / G1/S transition of mitotic cell cycle / Translesion Synthesis by POLH / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / cellular response to xenobiotic stimulus / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / response to estradiol / mitotic cell cycle / E3 ubiquitin ligases ubiquitinate target proteins / heart development / 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 / nuclear body / nucleotide binding / centrosome / chromatin binding / chromatin / protein-containing complex binding / 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: 3.6 Å
AuthorsRoske JJ / Yeeles JTP
Funding support United Kingdom, 1 items
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, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50222.png

Downloads & links

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Map

FileDownload / File: emd_50222.map.gz / Format: CCP4 / Size: 236.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 396 pix.
= 332.896 Å		0.84 Å/pix.
x 396 pix.
= 332.896 Å		0.84 Å/pix.
x 396 pix.
= 332.896 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84065 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.5552631 - 1.0578723
Average (Standard dev.)0.00019987496 (±0.01695038)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions396396396
Spacing396396396
CellA=B=C: 332.896 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50222_msk_1.map
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Mask #2

Fileemd_50222_msk_2.map
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Additional map: Map from focussed refinement with Mask around Polymerase...

Fileemd_50222_additional_1.map
AnnotationMap from focussed refinement with Mask around Polymerase epsilon catalytic domain
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Half map: #1

Fileemd_50222_half_map_1.map
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Half map: #2

Fileemd_50222_half_map_2.map
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Sample components

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Entire : Quaternary Complex of human leading strand polymerase epsilon, Pr...

EntireName: Quaternary Complex of human leading strand polymerase epsilon, Proliferating cell nuclear antigen (PCNA), substrate DNA and incoming nucleotide.
Components
  • Complex: Quaternary Complex of human leading strand polymerase epsilon, Proliferating cell nuclear antigen (PCNA), substrate DNA and incoming nucleotide.
    • Complex: DNA polymerase epsilon catalytic subunit A
      • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Complex: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
    • Complex: DNA
      • DNA: DNA nascent strand
      • DNA: DNA template strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: 2',3'-dideoxyadenosine triphosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Quaternary Complex of human leading strand polymerase epsilon, Pr...

SupramoleculeName: Quaternary Complex of human leading strand polymerase epsilon, Proliferating cell nuclear antigen (PCNA), substrate DNA and incoming nucleotide.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: DNA polymerase epsilon catalytic subunit A

SupramoleculeName: DNA polymerase epsilon catalytic subunit A / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Proliferating cell nuclear antigen

SupramoleculeName: Proliferating cell nuclear antigen / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)

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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: Trichoplusia ni (cabbage looper)
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(DE3) (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 nascent strand

MacromoleculeName: DNA nascent strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.074585 KDa
SequenceString:
(DC)(DC)(DT)(DT)(DC)(DC)(DA)(DC)(DT)(DT) (DC)(DC)(DC)(DA)(DA)(DC)(DC)(DC)(DT)(DC) (DA)(DC)(DC)(2DA)

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Macromolecule #4: DNA template strand

MacromoleculeName: DNA template strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.033732 KDa
SequenceString:
(DA)(DA)(DG)(DG)(DC)(DT)(DG)(DA)(DA)(DC) (DG)(DA)(DA)(DT)(DT)(DG)(DG)(DT)(DG)(DA) (DG)(DG)(DG)(DT)(DT)(DG)(DG)(DG)(DA) (DA)(DG)(DT)(DG)(DG)(DA)(DA)(DG)(DG)

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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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Macromolecule #6: 2',3'-dideoxyadenosine triphosphate

MacromoleculeName: 2',3'-dideoxyadenosine triphosphate / type: ligand / ID: 6 / Number of copies: 1 / Formula: DDS
Molecular weightTheoretical: 475.182 Da
Chemical component information

ChemComp-DDS:
2',3'-dideoxyadenosine triphosphate

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.08 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 175614
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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