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- EMDB-21701: Structure of the Saccharomyces cerevisiae polymerase epsilon holo... -

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Basic information

Entry
Database: EMDB / ID: EMD-21701
TitleStructure of the Saccharomyces cerevisiae polymerase epsilon holoenzyme
Map dataSaccharomyces cerevisiae polymerase epsilon holoenzyme
Sample
  • Complex: DNA polymerase epsilon
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: DNA polymerase epsilon subunit B
    • Protein or peptide: DNA polymerase epsilon subunit C
    • Protein or peptide: DNA polymerase epsilon subunit D
  • Ligand: ZINC ION
Function / homology
Function and homology information


: / : / chromatin remodeling => GO:0006338 / CHRAC / : / DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling ...: / : / chromatin remodeling => GO:0006338 / CHRAC / : / DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleosomal DNA binding / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / error-prone translesion synthesis / heterochromatin formation / base-excision repair, gap-filling / replication fork / base-excision repair / DNA-templated DNA replication / chromatin DNA binding / double-strand break repair via nonhomologous end joining / double-strand break repair / single-stranded DNA binding / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / cell cycle / protein heterodimerization activity / nucleotide binding / mRNA binding / DNA damage response / DNA binding / zinc ion binding / nucleus / cytoplasm
Similarity search - Function
DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain ...DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Histone-fold / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase epsilon catalytic subunit A / DNA polymerase epsilon subunit B / DNA polymerase epsilon subunit C / DNA polymerase epsilon subunit D
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYuan Z / Georgescu R / Schauer GD / O'Donnell M / Li H
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM131754 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM115809 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome.
Authors: Zuanning Yuan / Roxana Georgescu / Grant D Schauer / Michael E O'Donnell / Huilin Li /
Abstract: The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal ...The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ε revealing that the Dpb3-Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.
History
DepositionApr 14, 2020-
Header (metadata) releaseJul 8, 2020-
Map releaseJul 8, 2020-
UpdateJul 8, 2020-
Current statusJul 8, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wjv
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21701.png

Downloads & links

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Map

FileDownload / File: emd_21701.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSaccharomyces cerevisiae polymerase epsilon holoenzyme
Voxel sizeX=Y=Z: 1.029 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.048
Minimum - Maximum-0.11214168 - 0.22285458
Average (Standard dev.)0.0008572345 (±0.0064039514)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 263.424 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0291.0291.029
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z263.424263.424263.424
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1120.2230.001

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Supplemental data

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Sample components

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Entire : DNA polymerase epsilon

EntireName: DNA polymerase epsilon
Components
  • Complex: DNA polymerase epsilon
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: DNA polymerase epsilon subunit B
    • Protein or peptide: DNA polymerase epsilon subunit C
    • Protein or peptide: DNA polymerase epsilon subunit D
  • Ligand: ZINC ION

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Supramolecule #1: DNA polymerase epsilon

SupramoleculeName: DNA polymerase epsilon / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pRS

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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: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 255.992484 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MMFGKKKNNG GSSTARYSAG NKYNTLSNNY ALSAQQLLNA SKIDDIDSMM GFERYVPPQY NGRFDAKDID QIPGRVGWLT NMHATLVSQ ETLSSGSNGG GNSNDGERVT TNQGISGVDF YFLDEEGGSF KSTVVYDPYF FIACNDESRV NDVEELVKKY L ESCLKSLQ ...String:
MMFGKKKNNG GSSTARYSAG NKYNTLSNNY ALSAQQLLNA SKIDDIDSMM GFERYVPPQY NGRFDAKDID QIPGRVGWLT NMHATLVSQ ETLSSGSNGG GNSNDGERVT TNQGISGVDF YFLDEEGGSF KSTVVYDPYF FIACNDESRV NDVEELVKKY L ESCLKSLQ IIRKEDLTMD NHLLGLQKTL IKLSFVNSNQ LFEARKLLRP ILQDNANNNV QRNIYNVAAN GSEKVDAKHL IE DIREYDV PYHVRVSIDK DIRVGKWYKV TQQGFIEDTR KIAFADPVVM AFDIETTKPP LKFPDSAVDQ IMMISYMIDG EGF LITNRE IISEDIEDFE YTPKPEYPGF FTIFNENDEV ALLQRFFEHI RDVRPTVIST FNGDFFDWPF IHNRSKIHGL DMFD EIGFA PDAEGEYKSS YCSHMDCFRW VKRDSYLPQG SQGLKAVTQS KLGYNPIELD PELMTPYAFE KPQHLSEYSV SDAVA TYYL YMKYVHPFIF SLCTIIPLNP DETLRKGTGT LCEMLLMVQA YQHNILLPNK HTDPIERFYD GHLLESETYV GGHVES LEA GVFRSDLKNE FKIDPSAIDE LLQELPEALK FSVEVENKSS VDKVTNFEEI KNQITQKLLE LKENNIRNEL PLIYHVD VA SMYPNIMTTN RLQPDSIKAE RDCASCDFNR PGKTCARKLK WAWRGEFFPS KMDEYNMIKR ALQNETFPNK NKFSKKKV L TFDELSYADQ VIHIKKRLTE YSRKVYHRVK VSEIVEREAI VCQRENPFYV DTVKSFRDRR YEFKGLAKTW KGNLSKIDP SDKHARDEAK KMIVLYDSLQ LAHKVILNSF YGYVMRKGSR WYSMEMAGIT CLTGATIIQM ARALVERVGR PLELDTDGIW CILPKSFPE TYFFTLENGK KLYLSYPCSM LNYRVHQKFT NHQYQELKDP LNYIYETHSE NTIFFEVDGP YKAMILPSSK E EGKGIKKR YAVFNEDGSL AELKGFELKR RGELQLIKNF QSDIFKVFLE GDTLEGCYSA VASVCNRWLD VLDSHGLMLE DE DLVSLIC ENRSMSKTLK EYEGQKSTSI TTARRLGDFL GEDMVKDKGL QCKYIISSKP FNAPVTERAI PVAIFSADIP IKR SFLRRW TLDPSLEDLD IRTIIDWGYY RERLGSAIQK IITIPAALQG VSNPVPRVEH PDWLKRKIAT KEDKFKQTSL TKFF SKTKN VPTMGKIKDI EDLFEPTVEE DNAKIKIART TKKKAVSKRK RNQLTNEEDP LVLPSEIPSM DEDYVGWLNY QKIKW KIQA RDRKRRDQLF GNTNSSRERS ALGSMIRKQA ESYANSTWEV LQYKDSGEPG VLEVFVTING KVQNITFHIP KTIYMK FKS QTMPLQKIKN CLIEKSSASL PNNPKTSNPA GGQLFKITLP ESVFLEEKEN CTSIFNDENV LGVFEGTITP HQRAIMD LG ASVTFRSKAM GALGKGIQQG FEMKDLSMAE NERYLSGFSM DIGYLLHFPT SIGYEFFSLF KSWGDTITIL VLKPSNQA Q EINASSLGQI YKQMFEKKKG KIETYSYLVD IKEDINFEFV YFTDISKLYR RLSQETTKLK EERGLQFLLL LQSPFITKL LGTIRLLNQM PIVKLSLNEV LLPQLNWQPT LLKKLVNHVL SSGSWISHLI KLSQYSNIPI CNLRLDSMDY IIDVLYARKL KKENIVLWW NEKAPLPDHG GIQNDFDLNT SWIMNDSEFP KINNSGVYDN VVLDVGVDNL TVNTILTSAL INDAEGSDLV N NNMGIDDK DAVINSPSEF VHDAFSNDAL NVLRGMLKEW WDEALKENST ADLLVNSLAS WVQNPNAKLF DGLLRYHVHN LT KKALLQL VNEFSALGST IVYADRNQIL IKTNKYSPEN CYAYSQYMMK AVRTNPMFSY LDLNIKRYWD LLIWMDKFNF SGL ACIEIE EKENQDYTAV SQWQLKKFLS PIYQPEFEDW MMIILDSMLK TKQSYLKLNS GTQRPTQIVN VKKQDKEDSV ENSL NGFSH LFSKPLMKRV KKLFKNQQEF ILDPQYEADY VIPVLPGSHL NVKNPLLELV KSLCHVMLLS KSTILEIRTL RKELL KIFE LREFAKVAEF KDPSLSLVVP DFLCEYCFFI SDIDFCKAAP ESIFSCVRCH KAFNQVLLQE HLIQKLRSDI ESYLIQ DLR CSRCHKVKRD YMSAHCPCAG AWEGTLPRES IVQKLNVFKQ VAKYYGFDIL LSCIADLTI

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Macromolecule #2: DNA polymerase epsilon subunit B

MacromoleculeName: DNA polymerase epsilon subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 78.425852 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFGSGNVLPV KIQPPLLRPL AYRVLSRKYG LSIKSDGLSA LAEFVGTNIG ANWRQGPATI KFLEQFAAVW KQQERGLFID QSGVKEVIQ EMKEREKVEW SHEHPIQHEE NILGRTDDDE NNSDDEMPIA ADSSLQNVSL SSPMRQPTER DEYKQPFKPE S SKALDWRD ...String:
MFGSGNVLPV KIQPPLLRPL AYRVLSRKYG LSIKSDGLSA LAEFVGTNIG ANWRQGPATI KFLEQFAAVW KQQERGLFID QSGVKEVIQ EMKEREKVEW SHEHPIQHEE NILGRTDDDE NNSDDEMPIA ADSSLQNVSL SSPMRQPTER DEYKQPFKPE S SKALDWRD YFKVINASQQ QRFSYNPHKM QFIFVPNKKQ NGLGGIAGFL PDIEDKVQMF LTRYYLTNDR VMRNENFQNS DM FNPLSSM VSLQNELSNT NRQQQSSSMS ITPIKNLLGR DAQNFLLLGL LNKNFKGNWS LEDPSGSVEI DISQTIPTQG HYY VPGCMV LVEGIYYSVG NKFHVTSMTL PPGERREITL ETIGNLDLLG IHGISNNNFI ARLDKDLKIR LHLLEKELTD HKFV ILGAN LFLDDLKIMT ALSKILQKLN DDPPTLLIWQ GSFTSVPVFA SMSSRNISSS TQFKNNFDAL ATLLSRFDNL TENTT MIFI PGPNDLWGSM VSLGASGTLP QDPIPSAFTK KINKVCKNVV WSSNPTRIAY LSQEIVIFRD DLSGRFKRHR LEFPFN ESE DVYTENDNMM SKDTDIVPID ELVKEPDQLP QKVQETRKLV KTILDQGHLS PFLDSLRPIS WDLDHTLTLC PIPSTMV LC DTTSAQFDLT YNGCKVINPG SFIHNRRARY MEYVPSSKKT IQEEIYI

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Macromolecule #3: DNA polymerase epsilon subunit C

MacromoleculeName: DNA polymerase epsilon subunit C / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 22.694014 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSNLVKEKAP VFPISKVKKI AKCDPEYVIT SNVAISATAF AAELFVQNLV EESLVLAQLN SKGKTSLRLS LNSIEECVEK RDNFRFLED AIKQLKKNSA LDKKRELNMQ PGRSDQEVVI EEPELHEDDG VEEEEEEDEV SEEEEPVHNE ELLDDSKDQQ N DKSTRSVA ...String:
MSNLVKEKAP VFPISKVKKI AKCDPEYVIT SNVAISATAF AAELFVQNLV EESLVLAQLN SKGKTSLRLS LNSIEECVEK RDNFRFLED AIKQLKKNSA LDKKRELNMQ PGRSDQEVVI EEPELHEDDG VEEEEEEDEV SEEEEPVHNE ELLDDSKDQQ N DKSTRSVA SLLSRFQYKS ALDVGEHSDS SDIEVDHTKS TDP

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Macromolecule #4: DNA polymerase epsilon subunit D

MacromoleculeName: DNA polymerase epsilon subunit D / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 22.029484 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MPPKGWRKDA QGNYPTTSYI KEQENITIQD LLFPKSTIVN LAREVPQQSG KKLLINKDAS LALQRGATVF VNHLLLFARE IAKSQDKKS CSVDDVLSAL DHIGHSALKG PVRDKLDEYQ AAVEQRKKEK LDSGEVDADG DIDMGEDKEN VPVEKVKEHD E IEEQGDAL ...String:
MPPKGWRKDA QGNYPTTSYI KEQENITIQD LLFPKSTIVN LAREVPQQSG KKLLINKDAS LALQRGATVF VNHLLLFARE IAKSQDKKS CSVDDVLSAL DHIGHSALKG PVRDKLDEYQ AAVEQRKKEK LDSGEVDADG DIDMGEDKEN VPVEKVKEHD E IEEQGDAL QDVEESSEKK QKTESQDVET RVQNLEQT

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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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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 187298

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6wjv:
Structure of the Saccharomyces cerevisiae polymerase epsilon holoenzyme

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