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- PDB-7tid: Structure of the yeast clamp loader (Replication Factor C RFC) bo... -

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Entry
Database: PDB / ID: 7tid
TitleStructure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) and primer-template DNA
Components
  • (Replication factor C subunit ...) x 5
  • DNA (5'-D(*AP*GP*AP*CP*AP*CP*TP*AP*CP*GP*AP*GP*TP*AP*CP*AP*TP*A)-3')
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*AP*TP*GP*TP*AP*CP*TP*CP*GP*TP*AP*GP*TP*GP*TP*CP*T)-3')
  • Proliferating cell nuclear antigen
KeywordsREPLICATION/DNA / sliding clamp / DNA replication / AAA+ / clamp loader / REPLICATION-DNA complex
Function / homology
Function and homology information


DNA clamp unloading / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Processive synthesis on the lagging strand / Elg1 RFC-like complex / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA replication factor C complex ...DNA clamp unloading / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Processive synthesis on the lagging strand / Elg1 RFC-like complex / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA replication factor C complex / E3 ubiquitin ligases ubiquitinate target proteins / Polymerase switching / positive regulation of DNA metabolic process / SUMOylation of DNA replication proteins / DNA clamp loader activity / maintenance of DNA trinucleotide repeats / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / PCNA complex / Translesion Synthesis by POLH / DNA replication checkpoint signaling / establishment of mitotic sister chromatid cohesion / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / lagging strand elongation / postreplication repair / silent mating-type cassette heterochromatin formation / sister chromatid cohesion / mitotic sister chromatid cohesion / error-free translesion synthesis / leading strand elongation / DNA polymerase processivity factor activity / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / subtelomeric heterochromatin formation / mismatch repair / translesion synthesis / positive regulation of DNA repair / DNA damage checkpoint signaling / positive regulation of DNA replication / replication fork / nucleotide-excision repair / DNA-templated DNA replication / mitotic cell cycle / chromosome, telomeric region / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / nucleus / cytosol
Similarity search - Function
Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal domain / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : ...Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal domain / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / 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 / : / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Proliferating cell nuclear antigen / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 1 / Replication factor C subunit 4 / Replication factor C subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGaubitz, C. / Liu, X. / Pajak, J. / Stone, N. / Hayes, J. / Demo, G. / Kelch, B.A.
Funding support United States, Switzerland, Czech Republic, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM127776 United States
Swiss National Science Foundation168972 Switzerland
Swiss National Science Foundation177859 Switzerland
Ministry of Education (MoE, Czech Republic)LL2008 Czech Republic
CitationJournal: Elife / Year: 2022
Title: Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.
Authors: Christl Gaubitz / Xingchen Liu / Joshua Pajak / Nicholas P Stone / Janelle A Hayes / Gabriel Demo / Brian A Kelch /
Abstract: Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. ...Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.
History
DepositionJan 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_DOI ..._citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 28, 2024Group: Author supporting evidence / Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_audit_support / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_audit_support.country

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Replication factor C subunit 1
B: Replication factor C subunit 4
C: Replication factor C subunit 3
D: Replication factor C subunit 2
E: Replication factor C subunit 5
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Proliferating cell nuclear antigen
I: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*AP*TP*GP*TP*AP*CP*TP*CP*GP*TP*AP*GP*TP*GP*TP*CP*T)-3')
J: DNA (5'-D(*AP*GP*AP*CP*AP*CP*TP*AP*CP*GP*AP*GP*TP*AP*CP*AP*TP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)355,79519
Polymers353,17810
Non-polymers2,6179
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Replication factor C subunit ... , 5 types, 5 molecules ABCDE

#1: Protein Replication factor C subunit 1 / Replication factor C1 / Activator 1 95 kDa subunit / Cell division control protein 44


Mass: 95048.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC1, CDC44, YOR217W, YOR50-7 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P38630
#2: Protein Replication factor C subunit 4 / Replication factor C4 / Activator 1 37 kDa subunit


Mass: 36201.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC4, YOL094C, O0923 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P40339
#3: Protein Replication factor C subunit 3 / Replication factor C3 / Activator 1 40 kDa subunit


Mass: 38254.543 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC3, YNL290W, N0533 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P38629
#4: Protein Replication factor C subunit 2 / Replication factor C2 / Activator 1 41 kDa subunit


Mass: 39794.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC2, YJR068W, J1808 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P40348
#5: Protein Replication factor C subunit 5 / Replication factor C5 / Activator 1 40 kDa subunit


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, YBR087W, YBR0810 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P38251

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Protein , 1 types, 3 molecules FGH

#6: Protein Proliferating cell nuclear antigen / PCNA


Mass: 29525.713 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: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P15873

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DNA chain , 2 types, 2 molecules IJ

#7: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*AP*TP*GP*TP*AP*CP*TP*CP*GP*TP*AP*GP*TP*GP*TP*CP*T)-3')


Mass: 9172.891 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain DNA (5'-D(*AP*GP*AP*CP*AP*CP*TP*AP*CP*GP*AP*GP*TP*AP*CP*AP*TP*A)-3')


Mass: 6136.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 9 molecules

#9: 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.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


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

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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

ComponentName: yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) and primer-template DNA
Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.351 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2300 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_3699: / Classification: refinement
EM software
IDNameCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76270 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1SXJ

1sxj


Accession code: 1SXJ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221717
ELECTRON MICROSCOPYf_angle_d0.45829543
ELECTRON MICROSCOPYf_dihedral_angle_d14.8343209
ELECTRON MICROSCOPYf_chiral_restr0.0383440
ELECTRON MICROSCOPYf_plane_restr0.0033603

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