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- PDB-7kc0: Structure of the Saccharomyces cerevisiae replicative polymerase ... -

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Basic information

Entry
Database: PDB / ID: 7kc0
TitleStructure of the Saccharomyces cerevisiae replicative polymerase delta in complex with a primer/template and the PCNA clamp
Components
  • DNA (25-MER)
  • DNA (5'-D(P*AP*TP*GP*AP*CP*CP*AP*TP*GP*AP*TP*TP*AP*CP*GP*AP*AP*TP*TP*GP*C)-3')
  • DNA polymerase
  • POL31 isoform 1
  • POL32 isoform 1
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / Polymerase delta / PCNA / primed DNA / complex
Function / homology
Function and homology information


delta DNA polymerase complex / DNA-templated DNA replication maintenance of fidelity / DNA amplification / RNA-templated DNA biosynthetic process / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / meiotic mismatch repair / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / E3 ubiquitin ligases ubiquitinate target proteins / Polymerase switching ...delta DNA polymerase complex / DNA-templated DNA replication maintenance of fidelity / DNA amplification / RNA-templated DNA biosynthetic process / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / meiotic mismatch repair / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / E3 ubiquitin ligases ubiquitinate target proteins / Polymerase switching / SUMOylation of DNA replication proteins / positive regulation of DNA metabolic process / DNA replication, removal of RNA primer / maintenance of DNA trinucleotide repeats / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / establishment of mitotic sister chromatid cohesion / PCNA complex / Termination of translesion DNA synthesis / lagging strand elongation / double-strand break repair via break-induced replication / postreplication repair / silent mating-type cassette heterochromatin formation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / 3'-5'-DNA exonuclease activity / mitotic sister chromatid cohesion / DNA metabolic process / DNA strand elongation involved in DNA replication / error-free translesion synthesis / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / Dual incision in TC-NER / subtelomeric heterochromatin formation / mismatch repair / translesion synthesis / positive regulation of DNA repair / base-excision repair, gap-filling / replication fork / positive regulation of DNA replication / nucleotide-excision repair / base-excision repair / DNA-templated DNA replication / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / DNA replication / chromosome, telomeric region / molecular adaptor activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / identical protein binding / nucleus / metal ion binding / cytosol
Similarity search - Function
DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site ...DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / 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 signature. / DNA-directed DNA polymerase, family B, conserved site / : / 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 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / BJ4_G0010060.mRNA.1.CDS.1 / POL31 isoform 1 / DNA polymerase / Proliferating cell nuclear antigen / DNA polymerase delta catalytic subunit / Proliferating cell nuclear antigen ...2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / BJ4_G0010060.mRNA.1.CDS.1 / POL31 isoform 1 / DNA polymerase / Proliferating cell nuclear antigen / DNA polymerase delta catalytic subunit / Proliferating cell nuclear antigen / DNA polymerase delta small subunit / DNA polymerase delta subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZheng, F. / Georgescu, R. / Li, H. / O'Donnell, M.E.
Funding support United States, 3items
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)GM115809 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.
Authors: Fengwei Zheng / Roxana E Georgescu / Huilin Li / Michael E O'Donnell /
Abstract: The DNA polymerase (Pol) δ of (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and ...The DNA polymerase (Pol) δ of (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Å cryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.
History
DepositionOct 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
P: DNA (5'-D(P*AP*TP*GP*AP*CP*CP*AP*TP*GP*AP*TP*TP*AP*CP*GP*AP*AP*TP*TP*GP*C)-3')
T: DNA (25-MER)
F: Proliferating cell nuclear antigen
A: DNA polymerase
B: POL31 isoform 1
C: POL32 isoform 1
E: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)327,56113
Polymers326,6298
Non-polymers9325
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25040 Å2
ΔGint-141 kcal/mol
Surface area103540 Å2

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Components

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DNA chain , 2 types, 2 molecules PT

#1: DNA chain DNA (5'-D(P*AP*TP*GP*AP*CP*CP*AP*TP*GP*AP*TP*TP*AP*CP*GP*AP*AP*TP*TP*GP*C)-3')


Mass: 7681.985 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#2: DNA chain DNA (25-MER)


Mass: 11677.539 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Protein , 4 types, 6 molecules FEGABC

#3: Protein Proliferating cell nuclear antigen


Mass: 28944.051 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PCNA, POL30, GI526_G0000296, PACBIOSEQ_LOCUS349, PACBIOSEQ_LOCUS352, PACBIOSEQ_LOCUS359, PACBIOSEQ_LOCUS364, PACBIOSEQ_LOCUS365, PACBIOSEQ_LOCUS366, PACBIOSEQ_LOCUS371
Production host: Escherichia coli (E. coli) / References: UniProt: A0A6B7JGY6, UniProt: P15873*PLUS
#4: Protein DNA polymerase


Mass: 124707.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL3, GI526_G0000818 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A6A5Q0V0, UniProt: P15436*PLUS, DNA-directed DNA polymerase
#5: Protein POL31 isoform 1


Mass: 55352.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL31, GI526_G0003241 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5PTG9, UniProt: P46957*PLUS
#6: Protein POL32 isoform 1 / HLJ1_G0030030.mRNA.1.CDS.1 / BJ4_G0029460.mRNA.1.CDS.1


Mass: 40377.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL32, GI526_G0003272, PACBIOSEQ_LOCUS3556, PACBIOSEQ_LOCUS3579
Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5PT00, UniProt: P47110*PLUS

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Non-polymers , 4 types, 5 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-D3T / 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE


Type: DNA OH 3 prime terminus / Mass: 466.169 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O13P3
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Pol delta-PCNA-DNACOMPLEX#1-#60MULTIPLE SOURCES
2dsDNACOMPLEX#1-#21NATURAL
3ProteinsCOMPLEX#3-#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 68 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 133468 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319992
ELECTRON MICROSCOPYf_angle_d0.49527236
ELECTRON MICROSCOPYf_dihedral_angle_d20.5682953
ELECTRON MICROSCOPYf_chiral_restr0.0413112
ELECTRON MICROSCOPYf_plane_restr0.0033314

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