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- PDB-6s2f: Cryo-EM structure of Ctf18-1-8 in complex with the catalytic doma... -

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Basic information

Entry
Database: PDB / ID: 6s2f
TitleCryo-EM structure of Ctf18-1-8 in complex with the catalytic domain of DNA polymerase epsilon (Class 2)
Components
  • Chromosome transmission fidelity protein 18
  • Chromosome transmission fidelity protein 8
  • DNA polymerase epsilon catalytic subunit A
  • Sister chromatid cohesion protein DCC1
KeywordsREPLICATION / DNA polymerase / PCNA loader / protein complex
Function / homology
Function and homology information


maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / Ctf18 RFC-like complex / nucleotide-excision repair, DNA gap filling / SUMO binding / maintenance of DNA trinucleotide repeats / DNA replication proofreading / Activation of the pre-replicative complex ...maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / Ctf18 RFC-like complex / nucleotide-excision repair, DNA gap filling / SUMO binding / maintenance of DNA trinucleotide repeats / 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 / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / 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 / DNA-templated DNA replication / 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 replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / 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 / 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 ...Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / 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 / 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 / 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 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsGrabarczyk, D.B. / Song, B.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research FoundationGR 5152/3-1 Germany
CitationJournal: Nucleic Acids Res / Year: 2020
Title: Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication.
Authors: Katy Stokes / Alicja Winczura / Boyuan Song / Giacomo De Piccoli / Daniel B Grabarczyk /
Abstract: The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA ...The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.
History
DepositionJun 20, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 20, 2021Group: Database references / Category: citation / citation_author
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 / _citation_author.name

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)203,9325
Polymers203,5804
Non-polymers3521
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12570 Å2
ΔGint-71 kcal/mol
Surface area72780 Å2
MethodPISA

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Components

#1: Protein DNA polymerase epsilon catalytic subunit A / DNA polymerase II subunit A


Mass: 140397.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: POL2, DUN2, YNL262W, N0825 / Production host: Escherichia coli (E. coli) / References: UniProt: P21951, DNA-directed DNA polymerase
#2: Protein Chromosome transmission fidelity protein 8


Mass: 15189.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: CTF8, YHR191C / Production host: Escherichia coli (E. coli) / References: UniProt: P38877
#3: Protein/peptide Chromosome transmission fidelity protein 18


Mass: 3859.329 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: CTF18, CHL12, YMR078C, YM9582.03C / Production host: Escherichia coli (E. coli) / References: UniProt: P49956
#4: 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 (strain ATCC 204508 / S288c) (yeast)
Gene: DCC1, YCL016C, YCL16C / Production host: Escherichia coli (E. coli) / References: UniProt: P25559
#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


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

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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: Complex of the catalytic domain of DNA polymerase epsilon with the Ctf18-1-8 module of Ctf18-RFC
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
220 mMHEPES1
31 mMTCEP1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 75 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
PHENIX1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.14-3260model refinement
10RELION3.0 betainitial Euler assignment
11RELION3.0 betafinal Euler assignment
12RELION3.0 betaclassification
13RELION3.0 beta3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24967 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-ID
14M8O

4m8o

1
25OKC

5okc

1
RefinementHighest resolution: 5.8 Å / Stereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009412739
ELECTRON MICROSCOPYf_angle_d1.67317220
ELECTRON MICROSCOPYf_chiral_restr0.08611892
ELECTRON MICROSCOPYf_plane_restr0.012200
ELECTRON MICROSCOPYf_dihedral_angle_d7.48057727

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