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- PDB-8twa: Cryo-EM structure of S. cerevisiae Ctf18-RFC-PCNA-PolE-DNA complex -

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Basic information

Entry
Database: PDB / ID: 8twa
TitleCryo-EM structure of S. cerevisiae Ctf18-RFC-PCNA-PolE-DNA complex
Components
  • (Chromosome transmission fidelity protein ...) x 3
  • (Replication factor C subunit ...) x 4
  • DNA polymerase epsilon catalytic subunit A
  • Primer DNA
  • Proliferating cell nuclear antigen
  • Sister chromatid cohesion protein DCC1
  • Template DNA
KeywordsDNA BINDING PROTEIN/DNA / Ctf18 / RFC2-5 / PCNA / Pol2 / DNA / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / epsilon DNA polymerase complex / Processive synthesis on the lagging strand ...maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / epsilon DNA polymerase complex / Processive synthesis on the lagging strand / Elg1 RFC-like complex / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA replication factor C complex / telomere tethering at nuclear periphery / E3 ubiquitin ligases ubiquitinate target proteins / Polymerase switching / positive regulation of DNA metabolic process / SUMOylation of DNA replication proteins / maintenance of DNA trinucleotide repeats / SUMO binding / Activation of the pre-replicative complex / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / PCNA complex / DNA replication checkpoint signaling / nucleotide-excision repair, DNA gap filling / single-stranded DNA 3'-5' DNA exonuclease activity / establishment of mitotic sister chromatid cohesion / Activation of ATR in response to replication stress / DNA replication proofreading / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / lagging strand elongation / postreplication repair / mitotic intra-S DNA damage checkpoint signaling / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / mitotic sister chromatid cohesion / error-free translesion synthesis / DNA polymerase processivity factor activity / leading strand elongation / nuclear replication fork / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / chromosome, centromeric region / subtelomeric heterochromatin formation / DNA replication initiation / mismatch repair / translesion synthesis / error-prone translesion synthesis / base-excision repair, gap-filling / positive regulation of DNA repair / DNA damage checkpoint signaling / positive regulation of DNA replication / replication fork / nucleotide-excision repair / double-strand break repair via homologous recombination / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA replication / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / nucleotide binding / mRNA binding / chromatin / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / cytosol
Similarity search - Function
Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / 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 ...Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / 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 / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / 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, 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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / Proliferating cell nuclear antigen / DNA polymerase epsilon catalytic subunit A / Sister chromatid cohesion protein DCC1 / Replication factor C subunit 5 / Replication factor C subunit 3 ...ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / Proliferating cell nuclear antigen / DNA polymerase epsilon catalytic subunit A / Sister chromatid cohesion protein DCC1 / Replication factor C subunit 5 / Replication factor C subunit 3 / Chromosome transmission fidelity protein 8 / Replication factor C subunit 4 / Replication factor C subunit 2 / 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: 4.1 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

Downloads & links

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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
4: Replication factor C subunit 4
3: Replication factor C subunit 3
2: Replication factor C subunit 2
5: Replication factor C subunit 5
1: Chromosome transmission fidelity protein 18
X: Proliferating cell nuclear antigen
Y: Proliferating cell nuclear antigen
Z: Proliferating cell nuclear antigen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)650,25522
Polymers647,83314
Non-polymers2,4218
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 ... , 3 types, 3 molecules CD1

#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
#11: Protein Chromosome transmission fidelity protein 18


Mass: 84487.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF18, CHL12, YMR078C, YM9582.03C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P49956

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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 , 3 types, 5 molecules EBXYZ

#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
#12: Protein Proliferating cell nuclear antigen / PCNA


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: POL30, YBR088C, YBR0811 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P15873

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Replication factor C subunit ... , 4 types, 4 molecules 4325

#7: Protein Replication factor C subunit 4


Mass: 35782.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC4 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40339
#8: Protein Replication factor C subunit 3


Mass: 36818.879 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC3 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38629
#9: Protein Replication factor C subunit 2


Mass: 38256.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40348
#10: Protein Replication factor C subunit 5


Mass: 39993.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251

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Non-polymers , 4 types, 8 molecules

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


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#14: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#15: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#16: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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-PolE-DNA complex / Type: COMPLEX / Entity ID: #1-#12 / 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: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87643 / Symmetry type: POINT

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