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- EMDB-50228: Human DNA Polymerase epsilon bound to T-C mismatched DNA (Mismatc... -

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Basic information

Entry
Database: EMDB / ID: EMD-50228
TitleHuman DNA Polymerase epsilon bound to T-C mismatched DNA (Mismatch Excision state)
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: DNA
      • DNA: DNA nascent strand
      • DNA: DNA template strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: CALCIUM ION
KeywordsDNA / polymerase / epsilon / PCNA / leading strand / human / replication / replisome / proofreading
Function / homology
Function and homology information


DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA synthesis involved in DNA repair / leading strand elongation / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex ...DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA synthesis involved in DNA repair / leading strand elongation / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / embryonic organ development / base-excision repair, gap-filling / 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 / G1/S transition of mitotic cell cycle / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / nucleotide binding / chromatin binding / DNA binding / zinc ion binding / nucleoplasm / 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 / DNA polymerase family B, thumb domain ...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 / 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
DNA polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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_50228.png

Downloads & links

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Map

FileDownload / File: emd_50228.map.gz / Format: CCP4 / Size: 45.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.46 Å/pix.
x 228 pix.
= 332.88 Å		1.46 Å/pix.
x 228 pix.
= 332.88 Å		1.46 Å/pix.
x 228 pix.
= 332.88 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.46 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-2.6219466 - 6.3607664
Average (Standard dev.)0.0027646301 (±0.10105526)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions228228228
Spacing228228228
CellA=B=C: 332.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50228_msk_1.map
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Additional map: unsharpened map

Fileemd_50228_additional_1.map
Annotationunsharpened map
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Half map: #2

Fileemd_50228_half_map_1.map
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Half map: #1

Fileemd_50228_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: DNA
      • DNA: DNA nascent strand
      • DNA: DNA template strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: CALCIUM 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-#3

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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: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
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: 138.239609 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 DPVVLAFDIE 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: DNA nascent strand

MacromoleculeName: DNA nascent strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.489113 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DA)(DT) (DC)(DT)(DG)(DA)(DA)(DG)(DT)(DT)(DC)(DG) (DA)(DA)(DT)(DC)(DC)(DT)(DG)(DG)(DA) (DT)(DC)

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Macromolecule #3: DNA template strand

MacromoleculeName: DNA template strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.458104 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DA)(DT) (DC)(DC)(DA)(DG)(DG)(DA)(DT)(DT)(DC)(DG) (DA)(DA)(DC)(DT)(DT)(DC)(DA)(DG)(DA) (DT)(DC)

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Macromolecule #4: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 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: 36.4 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: 1.2 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: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 18858
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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