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- PDB-9b8r: Cryo-EM structure of S. cerevisiae PolE-core-DNA -

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Basic information

Entry
Database: PDB / ID: 9b8r
TitleCryo-EM structure of S. cerevisiae PolE-core-DNA
Components
  • DNA polymerase epsilon catalytic subunit
  • Primer DNA
  • Template DNA
KeywordsDNA BINDING PROTEIN/DNA / Pol2 / DNA / REPLICATION / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


epsilon DNA polymerase complex / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / DNA repair / nucleotide binding / DNA binding / zinc ion binding
Similarity search - Function
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-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily ...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-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B / 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
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA polymerase epsilon catalytic subunit
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 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
DepositionMar 31, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Oct 23, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
P: Primer DNA
T: Template DNA
E: DNA polymerase epsilon catalytic subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,9574
Polymers140,6053
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

#1: DNA chain Primer DNA


Mass: 3026.977 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#2: DNA chain Template DNA


Mass: 3680.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#3: Protein DNA polymerase epsilon catalytic subunit


Mass: 133898.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL2, GI527_G0004851 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: A0A8H4BWE7, DNA-directed DNA polymerase
#4: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
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: PolE-core-DNA complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121833 / Symmetry type: POINT

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