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- PDB-7z6h: Structure of DNA-bound human RAD17-RFC clamp loader and 9-1-1 che... -

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Basic information

Entry
Database: PDB / ID: 7z6h
TitleStructure of DNA-bound human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp
Components
  • (Cell cycle checkpoint ...) x 2
  • (Replication factor C subunit ...) x 4
  • Checkpoint protein HUS1
  • Hairpin DNA
KeywordsDNA BINDING PROTEIN / DNA damage checkpoint / Rad17-RFC / 9-1-1
Function / homology
Function and homology information


meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / meiotic recombination checkpoint signaling / response to organophosphorus / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching ...meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / meiotic recombination checkpoint signaling / response to organophosphorus / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / double-stranded DNA 3'-5' DNA exonuclease activity / exodeoxyribonuclease III / DNA clamp loader activity / DNA replication checkpoint signaling / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage / regulation of phosphorylation / Polymerase switching on the C-strand of the telomere / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / embryo development ending in birth or egg hatching / DNA duplex unwinding / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / negative regulation of DNA replication / Presynaptic phase of homologous DNA pairing and strand exchange / PCNA-Dependent Long Patch Base Excision Repair / ATP-dependent activity, acting on DNA / Activation of ATR in response to replication stress / response to UV / substantia nigra development / 3'-5' exonuclease activity / telomere maintenance / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / DNA damage checkpoint signaling / cellular response to ionizing radiation / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / regulation of protein phosphorylation / Translesion Synthesis by POLH / double-strand break repair via homologous recombination / G2/M DNA damage checkpoint / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / histone deacetylase binding / SH3 domain binding / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / intrinsic apoptotic signaling pathway in response to DNA damage / chromosome / site of double-strand break / Processing of DNA double-strand break ends / DNA replication / Regulation of TP53 Activity through Phosphorylation / damaged DNA binding / intracellular membrane-bounded organelle / DNA repair / DNA damage response / chromatin binding / nucleolus / protein kinase binding / enzyme binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Cell cycle checkpoint protein, Rad1 / Cell cycle checkpoint, Hus1 / Rad9 / Rad9 / Checkpoint protein Hus1/Mec3 / Hus1-like protein / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 ...Cell cycle checkpoint protein, Rad1 / Cell cycle checkpoint, Hus1 / Rad9 / Rad9 / 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 / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Cell cycle checkpoint protein RAD1 / Checkpoint protein HUS1 / Cell cycle checkpoint protein RAD17 / Replication factor C subunit 4 / Replication factor C subunit 2 / Replication factor C subunit 5 / Replication factor C subunit 3 / Cell cycle checkpoint control protein RAD9A
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsDay, M. / Oliver, A.W. / Pearl, L.H.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Cancer Research UKC302/A14532 United Kingdom
Cancer Research UKC302/A24386 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction.
Authors: Matthew Day / Antony W Oliver / Laurence H Pearl /
Abstract: The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or ...The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5'-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5' DNA end and fixes the orientation of 9-1-1 on the ssDNA.
History
DepositionMar 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 4, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 31, 2022Group: Database references / Category: citation
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cell cycle checkpoint control protein RAD9A
B: Cell cycle checkpoint protein RAD1,Cell cycle checkpoint protein RAD17
C: Checkpoint protein HUS1
D: Replication factor C subunit 4
E: Replication factor C subunit 3
F: Replication factor C subunit 2
G: Replication factor C subunit 5
K: Cell cycle checkpoint protein RAD1,Cell cycle checkpoint protein RAD17
X: Hairpin DNA
Y: Hairpin DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)516,74815
Polymers514,13210
Non-polymers2,6165
Water00
1


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

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Components

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Cell cycle checkpoint ... , 2 types, 3 molecules ABK

#1: Protein Cell cycle checkpoint control protein RAD9A / hRAD9 / DNA repair exonuclease rad9 homolog A


Mass: 44260.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD9A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q99638, exodeoxyribonuclease III
#2: Protein Cell cycle checkpoint protein RAD1,Cell cycle checkpoint protein RAD17 / hRAD1 / DNA repair exonuclease rad1 homolog / Rad1-like DNA damage checkpoint protein / hRad17 / RF- ...hRAD1 / DNA repair exonuclease rad1 homolog / Rad1-like DNA damage checkpoint protein / hRad17 / RF-C/activator 1 homolog


Mass: 115195.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD1, REC1, RAD17, R24L / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: O60671, UniProt: O75943, exodeoxyribonuclease III

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

#4: Protein Replication factor C subunit 4 / Activator 1 37 kDa subunit / A1 37 kDa subunit / Activator 1 subunit 4 / Replication factor C 37 ...Activator 1 37 kDa subunit / A1 37 kDa subunit / Activator 1 subunit 4 / Replication factor C 37 kDa subunit / RF-C 37 kDa subunit / RFC37


Mass: 39719.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RFC4 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P35249
#5: Protein Replication factor C subunit 3 / Activator 1 38 kDa subunit / A1 38 kDa subunit / Activator 1 subunit 3 / Replication factor C 38 ...Activator 1 38 kDa subunit / A1 38 kDa subunit / Activator 1 subunit 3 / Replication factor C 38 kDa subunit / RF-C 38 kDa subunit / RFC38


Mass: 40614.332 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RFC3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P40938
#6: Protein Replication factor C subunit 2 / Activator 1 40 kDa subunit / A1 40 kDa subunit / Activator 1 subunit 2 / Replication factor C 40 ...Activator 1 40 kDa subunit / A1 40 kDa subunit / Activator 1 subunit 2 / Replication factor C 40 kDa subunit / RF-C 40 kDa subunit / RFC40


Mass: 39203.207 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RFC2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P35250
#7: Protein Replication factor C subunit 5 / Activator 1 36 kDa subunit / A1 36 kDa subunit / Activator 1 subunit 5 / Replication factor C 36 ...Activator 1 36 kDa subunit / A1 36 kDa subunit / Activator 1 subunit 5 / Replication factor C 36 kDa subunit / RF-C 36 kDa subunit / RFC36


Mass: 40688.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RFC5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P40937

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Protein / DNA chain / Non-polymers , 3 types, 8 molecules CXY

#3: Protein Checkpoint protein HUS1 / hHUS1


Mass: 31731.854 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HUS1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O60921
#8: DNA chain Hairpin DNA


Mass: 23761.232 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: DNA with the sequence CCCGTATATTCTCCTACAGCACTAAATAATAGTGCTGTAGGAGAATATACGGGCTGCTCGTGTTGACAAGTACTGAT which forms a hairpin DNA molecule with 24 base-pairs of fully-complementary dsDNA capped ...Details: DNA with the sequence CCCGTATATTCTCCTACAGCACTAAATAATAGTGCTGTAGGAGAATATACGGGCTGCTCGTGTTGACAAGTACTGAT which forms a hairpin DNA molecule with 24 base-pairs of fully-complementary dsDNA capped with a tetraloop at one end, and with a 24 nucleotide 3' overhang
Source: (synth.) synthetic construct (others)
#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.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*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: DNA-bound human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 0.21 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE

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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: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 34.9 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150626 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00321048
ELECTRON MICROSCOPYf_angle_d0.59328586
ELECTRON MICROSCOPYf_dihedral_angle_d12.7543037
ELECTRON MICROSCOPYf_chiral_restr0.043320
ELECTRON MICROSCOPYf_plane_restr0.0053509

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