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- PDB-8rjw: Human RAD52 open ring - ssDNA complex -

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Basic information

Entry
Database: PDB / ID: 8rjw
TitleHuman RAD52 open ring - ssDNA complex
Components
  • DNA repair protein RAD52 homolog
  • ssDNADNA
KeywordsDNA BINDING PROTEIN / ssDNA binding protein / DNA damage repair / Single-strand annealing
Function / homology
Function and homology information


double-strand break repair via single-strand annealing / DNA double-strand break processing involved in repair via single-strand annealing / DNA recombinase assembly / mitotic recombination / regulation of nucleotide-excision repair / HDR through MMEJ (alt-NHEJ) / HDR through Single Strand Annealing (SSA) / SUMOylation of DNA damage response and repair proteins / protein-DNA complex / double-strand break repair via homologous recombination ...double-strand break repair via single-strand annealing / DNA double-strand break processing involved in repair via single-strand annealing / DNA recombinase assembly / mitotic recombination / regulation of nucleotide-excision repair / HDR through MMEJ (alt-NHEJ) / HDR through Single Strand Annealing (SSA) / SUMOylation of DNA damage response and repair proteins / protein-DNA complex / double-strand break repair via homologous recombination / double-strand break repair / single-stranded DNA binding / cellular response to oxidative stress / DNA recombination / protein-containing complex / DNA binding / nucleoplasm / identical protein binding / nucleus
Similarity search - Function
DNA recombination/repair protein Rad52 / DNA repair protein Rad52/59/22 / Rad52 family / DNA repair protein Rad52/59/22 superfamily / Rad52/22 family double-strand break repair protein
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA repair protein RAD52 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsLiang, C.C. / West, S.C.
Funding support United Kingdom, European Union, Switzerland, 8items
OrganizationGrant numberCountry
The Francis Crick InstituteCC2098 United Kingdom
Cancer Research UKCC2098 United Kingdom
Medical Research Council (MRC, United Kingdom)CC2098 United Kingdom
Wellcome TrustCC2098 United Kingdom
European Research Council (ERC)ERC-ADG-666400European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W01355X/1 United Kingdom
Louis-Jeantet Foundation Switzerland
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Nature / Year: 2024
Title: Mechanism of single-stranded DNA annealing by RAD52-RPA complex.
Authors: Chih-Chao Liang / Luke A Greenhough / Laura Masino / Sarah Maslen / Ilirjana Bajrami / Marcel Tuppi / Mark Skehel / Ian A Taylor / Stephen C West /
Abstract: RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52 promotes the annealing of ...RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA) and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal, and aberrant expression of RAD52 is associated with poor cancer prognosis. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.
History
DepositionDec 21, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA repair protein RAD52 homolog
B: DNA repair protein RAD52 homolog
C: DNA repair protein RAD52 homolog
D: DNA repair protein RAD52 homolog
E: DNA repair protein RAD52 homolog
F: DNA repair protein RAD52 homolog
G: DNA repair protein RAD52 homolog
H: DNA repair protein RAD52 homolog
I: DNA repair protein RAD52 homolog
J: ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)423,16715
Polymers423,04510
Non-polymers1225
Water70339
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area34050 Å2
ΔGint-203 kcal/mol
Surface area55530 Å2

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Components

#1: Protein
DNA repair protein RAD52 homolog /


Mass: 46232.656 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD52 / Production host: Escherichia coli (E. coli) / References: UniProt: P43351
#2: DNA chain ssDNA / DNA


Mass: 6951.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 39 / Source method: isolated from a natural source / Formula: H2O
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 ring conformation of human RAD52 in complex with ssDNA
Type: COMPLEX
Details: Recombinant RAD52 purified from E. coli, and the open ring conformation was separated by cation exchange. The RAD52-ssDNA complex was reconstituted in vitro.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaClSodium chloride1
30.25 mMTCEPC9H15O6P1
40.5 %Octyl beta glucosideC14H28O61
52 mMMagnesium acetateMg(CH3COO)21
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2250 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13176589
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 587046 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8RJ3

8rj3


Accession code: 8RJ3 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 55.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004410455
ELECTRON MICROSCOPYf_angle_d0.503514124
ELECTRON MICROSCOPYf_chiral_restr0.0411510
ELECTRON MICROSCOPYf_plane_restr0.00281764
ELECTRON MICROSCOPYf_dihedral_angle_d15.47471589

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