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- PDB-8dqw: Open state of Rad24-RFC:9-1-1 bound to a 5' ss/dsDNA junction -

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Basic information

Entry
Database: PDB / ID: 8dqw
TitleOpen state of Rad24-RFC:9-1-1 bound to a 5' ss/dsDNA junction
Components
  • (DNA damage checkpoint control protein ...) x 2
  • (Replication factor C subunit ...) x 4
  • DDC1 isoform 1
  • DNA (5'-D(P*CP*GP*TP*CP*CP*CP*TP*TP*CP*C)-3')
  • DNA (50-MER)
  • RAD24 isoform 1
KeywordsREPLICATION/DNA / DNA damage checkpoint / REPLICATION-DNA complex
Function / homology
Function and homology information


meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / cell cycle / Rad17 RFC-like complex / : / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Polymerase switching / DNA clamp loader activity ...meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / cell cycle / Rad17 RFC-like complex / : / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Polymerase switching / DNA clamp loader activity / Translesion synthesis by REV1 / : / : / : / DNA replication checkpoint signaling / : / Activation of ATR in response to replication stress / mitotic DNA replication checkpoint signaling / telomere maintenance via recombination / mitotic intra-S DNA damage checkpoint signaling / reciprocal meiotic recombination / recombinational repair / sister chromatid cohesion / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / subtelomeric heterochromatin formation / mismatch repair / 3'-5' exonuclease activity / telomere maintenance / DNA damage checkpoint signaling / cellular response to ionizing radiation / meiotic cell cycle / nucleotide-excision repair / double-strand break repair via homologous recombination / DNA-templated DNA replication / double-strand break repair / site of double-strand break / double-stranded DNA binding / damaged DNA binding / chromosome, telomeric region / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Ddc1 / DNA damage checkpoint control protein Rad17 / Checkpoint protein Hus1/Mec3 / Hus1-like protein / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 / Repair protein Rad1/Rec1/Rad17 / Zinc Finger, Delta Prime; domain 3 - #10 ...Ddc1 / DNA damage checkpoint control protein Rad17 / Checkpoint protein Hus1/Mec3 / Hus1-like protein / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 / Repair protein Rad1/Rec1/Rad17 / Zinc Finger, Delta Prime; domain 3 - #10 / Zinc Finger, Delta Prime; domain 3 / RFC1-like, AAA+ ATPase lid 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 / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Helicase, Ruva Protein; domain 3 - #60 / : / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Up-down Bundle / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / DDC1 isoform 1 / DNA damage checkpoint control protein RAD17 / RAD24 isoform 1 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 ...PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / DDC1 isoform 1 / DNA damage checkpoint control protein RAD17 / RAD24 isoform 1 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 / Replication factor C subunit 2 / DNA damage checkpoint control protein MEC3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsSchrecker, M. / Hite, R.K.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM107239 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127428 United States
CitationJournal: Elife / Year: 2022
Title: Multistep loading of a DNA sliding clamp onto DNA by replication factor C.
Authors: Marina Schrecker / Juan C Castaneda / Sujan Devbhandari / Charanya Kumar / Dirk Remus / Richard K Hite /
Abstract: The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader ...The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.
History
DepositionJul 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Feb 14, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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: DNA damage checkpoint control protein RAD17
G: DDC1 isoform 1
H: DNA damage checkpoint control protein MEC3
A: RAD24 isoform 1
J: DNA (50-MER)
I: DNA (5'-D(P*CP*GP*TP*CP*CP*CP*TP*TP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)437,15019
Polymers434,51710
Non-polymers2,6339
Water7,728429
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: 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: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40339
#2: 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)
Strain: ATCC 204508 / S288c / Gene: RFC3, YNL290W, N0533 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38629
#3: 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: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40348
#4: 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: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251

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DNA damage checkpoint control protein ... , 2 types, 2 molecules FH

#5: Protein DNA damage checkpoint control protein RAD17


Mass: 45637.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD17, GI527_G0005737 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BW58
#7: Protein DNA damage checkpoint control protein MEC3


Mass: 53207.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: MEC3, PIP3, PSO9, YLR288C, L8003.15 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02574

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Protein , 2 types, 2 molecules GA

#6: Protein DDC1 isoform 1


Mass: 73850.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DDC1, GI527_G0005854 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BUG7
#8: Protein RAD24 isoform 1


Mass: 80096.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD24, GI527_G0001991 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H8UM36

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DNA chain , 2 types, 2 molecules JI

#9: DNA chain DNA (50-MER)


Mass: 21507.701 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#10: DNA chain DNA (5'-D(P*CP*GP*TP*CP*CP*CP*TP*TP*CP*C)-3')


Mass: 5972.841 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 4 types, 438 molecules

#11: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#12: 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
#13: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#14: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 429 / Source method: isolated from a natural source / Formula: H2O

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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: Open state of Rad24-RFC:9-1-1 bound to a 5' ss/dsDNA junction
Type: COMPLEX / Entity ID: #1, #3-#10 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES-KOH1
2300 mMpotassium acetate1
37 mMmagnesium acetate1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K

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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: 2000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
7PHENIXmodel fitting
12cryoSPARC3.33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 938420 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7ST9

7st9


Accession code: 7ST9 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00223662
ELECTRON MICROSCOPYf_angle_d0.4632137
ELECTRON MICROSCOPYf_dihedral_angle_d11.343427
ELECTRON MICROSCOPYf_chiral_restr0.0393694
ELECTRON MICROSCOPYf_plane_restr0.0033955

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