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- PDB-7sh2: Structure of the yeast Rad24-RFC loader bound to DNA and the open... -

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Basic information

Entry
Database: PDB / ID: 7sh2
TitleStructure of the yeast Rad24-RFC loader bound to DNA and the open 9-1-1 clamp
Components
  • (DNA damage checkpoint ...) x 2
  • (Replication factor C subunit ...) x 4
  • Checkpoint protein RAD24
  • Crick strand
  • Mitosis Entry Checkpoint protein MEC3
  • Watson strand
KeywordsDNA BINDING PROTEIN/DNA / DNA damage repair / Rad24-RFC / 9-1-1 clamp / DNA clamp / alternative clamp loader / DNA damage signaling / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


checkpoint clamp complex / meiotic recombination checkpoint signaling / DNA clamp unloading / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / Ctf18 RFC-like complex / DNA replication factor C complex / Polymerase switching / DNA clamp loader activity ...checkpoint clamp complex / meiotic recombination checkpoint signaling / DNA clamp unloading / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / Ctf18 RFC-like complex / DNA replication factor C complex / Polymerase switching / DNA clamp loader activity / nuclease activity / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Translesion Synthesis by POLH / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / reciprocal meiotic recombination / mitotic intra-S DNA damage checkpoint signaling / recombinational repair / sister chromatid cohesion / mitotic sister chromatid cohesion / leading strand elongation / protein kinase activator activity / mitotic G2 DNA damage checkpoint signaling / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / mismatch repair / mitotic G1 DNA damage checkpoint signaling / DNA damage checkpoint signaling / condensed nuclear chromosome / meiotic cell cycle / cellular response to ionizing radiation / nucleotide-excision repair / DNA-templated DNA replication / double-strand break repair / double-stranded DNA binding / damaged DNA binding / DNA repair / chromatin binding / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ddc1 / DNA damage checkpoint control protein Rad17 / Rad17 P-loop domain / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 / Repair protein Rad1/Rec1/Rad17 / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain ...Ddc1 / DNA damage checkpoint control protein Rad17 / Rad17 P-loop domain / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 / Repair protein Rad1/Rec1/Rad17 / 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 / : / 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) / : / Checkpoint protein RAD24 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 / Replication factor C subunit 2 ...ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / : / Checkpoint protein RAD24 / 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 RAD17 / DNA damage checkpoint protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsZheng, F. / Georgescu, R. / Yao, Y.N. / O'Donnell, M.E. / 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) United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp.
Authors: Fengwei Zheng / Roxana E Georgescu / Nina Y Yao / Michael E O'Donnell / Huilin Li /
Abstract: The 9-1-1 DNA checkpoint clamp is loaded onto 5'-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in ...The 9-1-1 DNA checkpoint clamp is loaded onto 5'-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate clamp loader in which Rad24 replaces Rfc1 in the RFC1-5 clamp loader of proliferating cell nuclear antigen (PCNA). The 9-1-1 clamp loading mechanism has been a mystery, because, unlike RFC, which loads PCNA onto a 3'-recessed junction, Rad24-RFC loads the 9-1-1 ring onto a 5'-recessed DNA junction. Here we report two cryo-EM structures of Rad24-RFC-DNA with a closed or 27-Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. Unlike RFC, which encircles DNA, Rad24 binds 5'-DNA on its surface, not inside the loader, and threads the 3' ssDNA overhang into the 9-1-1 clamp from above the ring.
History
DepositionOct 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 23, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 27, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Checkpoint protein RAD24
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: Mitosis Entry Checkpoint protein MEC3
G: DNA damage checkpoint control protein RAD17
H: DNA damage checkpoint protein DDC1
P: Watson strand
T: Crick strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)416,86819
Polymers414,25110
Non-polymers2,6179
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, verified by 2D and 3D classification of single particles
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AF

#1: Protein Checkpoint protein RAD24


Mass: 75823.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD24, YER173W, SYGP-ORF60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32641
#6: Protein Mitosis Entry Checkpoint protein MEC3 / DNA damage checkpoint control protein MEC3 / Y55_G0029450.mRNA.1.CDS.1


Mass: 50407.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS4067, PACBIOSEQ_LOCUS4215, SCNYR20_0004029900, SCP684_0004029500
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PTK1

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

#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: Saccharomyces cerevisiae (brewer's yeast) / 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: Saccharomyces cerevisiae (brewer's yeast) / 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: Saccharomyces cerevisiae (brewer's yeast) / 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: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251

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DNA damage checkpoint ... , 2 types, 2 molecules GH

#7: Protein DNA damage checkpoint control protein RAD17 / DNA repair exonuclease 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, YOR368W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P48581
#8: Protein DNA damage checkpoint protein DDC1


Mass: 69787.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DDC1, YPL194W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08949

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DNA chain , 2 types, 2 molecules PT

#9: DNA chain Watson strand


Mass: 6136.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#10: DNA chain Crick strand


Mass: 12214.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 9 molecules

#11: 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
#12: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#13: 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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Rad24-RFC-911 clamp-DNACOMPLEX#1-#100MULTIPLE SOURCES
2dsDNACOMPLEX#9-#101NATURALsynthetic DNA
3ProteinsCOMPLEX#1-#81RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12synthetic construct (others)32630
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 302403 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221332
ELECTRON MICROSCOPYf_angle_d0.54328919
ELECTRON MICROSCOPYf_dihedral_angle_d10.8412992
ELECTRON MICROSCOPYf_chiral_restr0.0413346
ELECTRON MICROSCOPYf_plane_restr0.0043560

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