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- PDB-8tw9: Cryo-EM structure of S. cerevisiae PolE-Ctf18-8-1-DNA -

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Basic information

Entry
Database: PDB / ID: 8tw9
TitleCryo-EM structure of S. cerevisiae PolE-Ctf18-8-1-DNA
Components
  • (Chromosome transmission fidelity protein ...) x 2
  • DNA polymerase epsilon catalytic subunit A
  • Primer DNA
  • Sister chromatid cohesion protein DCC1
  • Template DNA
KeywordsDNA BINDING PROTEIN/DNA / Pol2 / Ctf18-8-1 / DNA / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / telomere tethering at nuclear periphery / Ctf18 RFC-like complex / maintenance of DNA trinucleotide repeats / SUMO binding / nucleotide-excision repair, DNA gap filling / Activation of the pre-replicative complex ...maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / telomere tethering at nuclear periphery / Ctf18 RFC-like complex / maintenance of DNA trinucleotide repeats / SUMO binding / nucleotide-excision repair, DNA gap filling / Activation of the pre-replicative complex / DNA replication proofreading / Termination of translesion DNA synthesis / 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 / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / Dual incision in TC-NER / chromosome, centromeric region / DNA replication initiation / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / double-strand break repair via homologous recombination / base-excision repair / double-strand break repair via nonhomologous end joining / DNA-templated DNA replication / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / nucleotide binding / mRNA binding / chromatin / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / ATP binding / nucleus
Similarity search - Function
Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / RFC1-like, AAA+ ATPase lid domain / : / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / DNA polymerase epsilon, catalytic subunit A, C-terminal ...Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / RFC1-like, AAA+ ATPase lid domain / : / : / 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 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribonuclease H superfamily / Ribonuclease H-like superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA polymerase epsilon catalytic subunit A / Sister chromatid cohesion protein DCC1 / Chromosome transmission fidelity protein 8 / Chromosome transmission fidelity protein 18
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsYuan, Z. / Georgescu, R. / O'Donnell, M. / Li, H.
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)M.E.O. United States
CitationJournal: Science / Year: 2024
Title: Mechanism of PCNA loading by Ctf18-RFC for leading-strand DNA synthesis.
Authors: Zuanning Yuan / Roxana Georgescu / Nina Y Yao / Olga Yurieva / Michael E O'Donnell / Huilin Li /
Abstract: The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific ...The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Polε contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Polε to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Polε off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Polε. These elements in both Ctf18-RFC and Polε provide specificity in loading PCNA onto DNA for Polε.
History
DepositionAug 20, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Chromosome transmission fidelity protein 18
P: Primer DNA
T: Template DNA
E: DNA polymerase epsilon catalytic subunit A
D: Chromosome transmission fidelity protein 8
B: Sister chromatid cohesion protein DCC1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)326,0147
Polymers325,6626
Non-polymers3521
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Chromosome transmission fidelity protein ... , 2 types, 2 molecules CD

#1: Protein/peptide Chromosome transmission fidelity protein 18


Mass: 3171.607 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF18 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P49956
#5: Protein Chromosome transmission fidelity protein 8


Mass: 15058.493 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF8 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38877

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DNA chain , 2 types, 2 molecules PT

#2: DNA chain Primer DNA


Mass: 2737.795 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Template DNA


Mass: 4567.998 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein , 2 types, 2 molecules EB

#4: Protein DNA polymerase epsilon catalytic subunit A / 3'-5' exodeoxyribonuclease / DNA polymerase II subunit A


Mass: 255992.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL2, DUN2, YNL262W, N0825 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters
#6: Protein Sister chromatid cohesion protein DCC1 / Defective in sister chromatid cohesion protein 1


Mass: 44133.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DCC1, YCL016C, YCL16C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25559

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN
Has protein modificationN

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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

ComponentName: Ctf18-RFC-PCNA complex / Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 385806 / Symmetry type: POINT

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