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- EMDB-50224: Human DNA polymerase epsilon bound to DNA and PCNA (closed confor... -

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Basic information

Entry
Database: EMDB / ID: EMD-50224
TitleHuman DNA polymerase epsilon bound to DNA and PCNA (closed conformation)
Map data
Sample
  • Complex: Quaternary Complex of human leading strand polymerase epsilon, Proliferating cell nuclear antigen (PCNA), substrate DNA and incoming nucleotide.
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: DNA nascent strand
    • DNA: DNA template strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: 2',3'-dideoxyadenosine triphosphate
KeywordsDNA / polymerase / epsilon / PCNA / leading strand / human / replication / replisome / proofreading
Function / homology
Function and homology information


DNA replication initiation / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / epsilon DNA polymerase complex / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina ...DNA replication initiation / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / epsilon DNA polymerase complex / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / Polymerase switching / nucleotide-excision repair, DNA gap filling / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / PCNA complex / DNA replication proofreading / 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 / leading strand elongation / DNA polymerase processivity factor activity / 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 / estrous cycle / mismatch repair / embryonic organ development / translesion synthesis / response to cadmium ion / DNA polymerase binding / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / male germ cell nucleus / replication fork / nuclear estrogen receptor binding / liver regeneration / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / receptor tyrosine kinase binding / DNA-templated DNA replication / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / G1/S transition of mitotic cell cycle / cellular response to UV / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / mitotic cell cycle / heart development / 4 iron, 4 sulfur cluster binding / DNA replication / damaged DNA binding / DNA-directed DNA polymerase / chromosome, telomeric region / DNA-directed DNA polymerase activity / nuclear body / nucleotide binding / centrosome / chromatin binding / protein-containing complex binding / chromatin / negative regulation of transcription by RNA polymerase II / enzyme binding / DNA binding / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / 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 / Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA ...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: 3.75 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-
UpdateAug 21, 2024-
Current statusAug 21, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50224.png

Downloads & links

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Map

FileDownload / File: emd_50224.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: 0.11
Minimum - Maximum-0.38926312 - 0.69688785
Average (Standard dev.)0.00021045878 (±0.012566012)
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_50224_msk_1.map
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Additional map: #1

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Half map: #2

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Half map: #1

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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.
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: DNA nascent strand
    • DNA: DNA template strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: 2',3'-dideoxyadenosine triphosphate

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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
Source (natural)Organism: Homo sapiens (human)

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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: synthetic construct (others)
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: synthetic construct (others)
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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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: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 92356
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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