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- EMDB-19255: Human Replication protein A (RPA; trimeric core) - ssDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-19255
TitleHuman Replication protein A (RPA; trimeric core) - ssDNA complex
Map data
Sample
  • Complex: Open ring conformation of human RAD52 in complex with ssDNA
    • Protein or peptide: Replication protein A 70 kDa DNA-binding subunit, N-terminally processed
    • Protein or peptide: Replication protein A 32 kDa subunit
    • Protein or peptide: Replication protein A 14 kDa subunit
KeywordsssDNA binding protein / DNA damage repair / Single-strand annealing / DNA BINDING PROTEIN
Function / homology
Function and homology information


protein localization to chromosome / DNA replication factor A complex / lateral element / regulation of DNA damage checkpoint / single-stranded telomeric DNA binding / G-rich strand telomeric DNA binding / Removal of the Flap Intermediate / chromatin-protein adaptor activity / protein localization to site of double-strand break / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) ...protein localization to chromosome / DNA replication factor A complex / lateral element / regulation of DNA damage checkpoint / single-stranded telomeric DNA binding / G-rich strand telomeric DNA binding / Removal of the Flap Intermediate / chromatin-protein adaptor activity / protein localization to site of double-strand break / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Removal of the Flap Intermediate from the C-strand / HDR through Single Strand Annealing (SSA) / regulation of double-strand break repair via homologous recombination / telomeric DNA binding / Impaired BRCA2 binding to RAD51 / hemopoiesis / Presynaptic phase of homologous DNA pairing and strand exchange / site of DNA damage / PCNA-Dependent Long Patch Base Excision Repair / Regulation of HSF1-mediated heat shock response / Activation of the pre-replicative complex / HSF1 activation / telomere maintenance via telomerase / mismatch repair / Activation of ATR in response to replication stress / SUMOylation of DNA damage response and repair proteins / mitotic G1 DNA damage checkpoint signaling / homeostasis of number of cells within a tissue / telomere maintenance / regulation of mitotic cell cycle / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic cell cycle / male germ cell nucleus / nucleotide-excision repair / Fanconi Anemia Pathway / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / double-strand break repair via homologous recombination / base-excision repair / PML body / G2/M DNA damage checkpoint / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / Meiotic recombination / DNA-templated DNA replication / Formation of Incision Complex in GG-NER / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / single-stranded DNA binding / regulation of cell population proliferation / site of double-strand break / Processing of DNA double-strand break ends / protein phosphatase binding / DNA recombination / Regulation of TP53 Activity through Phosphorylation / in utero embryonic development / damaged DNA binding / chromosome, telomeric region / DNA replication / nuclear body / DNA repair / positive regulation of cell population proliferation / DNA damage response / ubiquitin protein ligase binding / chromatin binding / chromatin / enzyme binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain ...Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain / Replication protein A OB domain / : / Replication factor A, C-terminal / Replication factor-A C terminal domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Replication protein A 32 kDa subunit / Replication protein A 70 kDa DNA-binding subunit / Replication protein A 14 kDa subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiang CC / West SC
Funding support United Kingdom, European Union, Switzerland, 8 items
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 22, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19255.png

Downloads & links

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Map

FileDownload / File: emd_19255.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.009
Minimum - Maximum-0.028584117 - 0.052212548
Average (Standard dev.)0.000015986516 (±0.0006004926)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_19255_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19255_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Open ring conformation of human RAD52 in complex with ssDNA

EntireName: Open ring conformation of human RAD52 in complex with ssDNA
Components
  • Complex: Open ring conformation of human RAD52 in complex with ssDNA
    • Protein or peptide: Replication protein A 70 kDa DNA-binding subunit, N-terminally processed
    • Protein or peptide: Replication protein A 32 kDa subunit
    • Protein or peptide: Replication protein A 14 kDa subunit

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Supramolecule #1: Open ring conformation of human RAD52 in complex with ssDNA

SupramoleculeName: Open ring conformation of human RAD52 in complex with ssDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Replication protein A 70 kDa DNA-binding subunit, N-terminally pr...

MacromoleculeName: Replication protein A 70 kDa DNA-binding subunit, N-terminally processed
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.212977 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVGQLSEGAI AAIMQKGDTN IKPILQVINI RPITTGNSPP RYRLLMSDGL NTLSSFMLAT QLNPLVEEEQ LSSNCVCQIH RFIVNTLKD GRRVVILMEL EVLKSAEAVG VKIGNPVPYN EGLGQPQVAP PAPAASPAAS SRPQPQNGSS GMGSTVSKAY G ASKTFGKA ...String:
MVGQLSEGAI AAIMQKGDTN IKPILQVINI RPITTGNSPP RYRLLMSDGL NTLSSFMLAT QLNPLVEEEQ LSSNCVCQIH RFIVNTLKD GRRVVILMEL EVLKSAEAVG VKIGNPVPYN EGLGQPQVAP PAPAASPAAS SRPQPQNGSS GMGSTVSKAY G ASKTFGKA AGPSLSHTSG GTQSKVVPIA SLTPYQSKWT ICARVTNKSQ IRTWSNSRGE GKLFSLELVD ESGEIRATAF NE QVDKFFP LIEVNKVYYF SKGTLKIANK QFTAVKNDYE MTFNNETSVM PCEDDHHLPT VQFDFTGIDD LENKSKDSLV DII GICKSY EDATKITVRS NNREVAKRNI YLMDTSGKVV TATLWGEDAD KFDGSRQPVL AIKGARVSDF GGRSLSVLSS STII ANPDI PEAYKLRGWF DAEGQALDGV SISDLKSGGV GGSNTNWKTL YEVKSENLGQ GDKPDYFSSV ATVVYLRKEN CMYQA CPTQ DCNKKVIDQQ NGLYRCEKCD TEFPNFKYRM ILSVNIADFQ ENQWVTCFQE SAEAILGQNA AYLGELKDKN EQAFEE VFQ NANFRSFIFR VRVKVETYND ESRIKATVMD VKPVDYREYG RRLVMSIRRS ALM

UniProtKB: Replication protein A 70 kDa DNA-binding subunit

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Macromolecule #2: Replication protein A 32 kDa subunit

MacromoleculeName: Replication protein A 32 kDa subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.276795 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWNSGFESYG SSSYGGAGGY TQSPGGFGSP APSQAEKKSR ARAQHIVPCT ISQLLSATLV DEVFRIGNVE ISQVTIVGII RHAEKAPTN IVYKIDDMTA APMDVRQWVD TDDTSSENTV VPPETYVKVA GHLRSFQNKK SLVAFKIMPL EDMNEFTTHI L EVINAHMV ...String:
MWNSGFESYG SSSYGGAGGY TQSPGGFGSP APSQAEKKSR ARAQHIVPCT ISQLLSATLV DEVFRIGNVE ISQVTIVGII RHAEKAPTN IVYKIDDMTA APMDVRQWVD TDDTSSENTV VPPETYVKVA GHLRSFQNKK SLVAFKIMPL EDMNEFTTHI L EVINAHMV LSKANSQPSA GRAPISNPGM SEAGNFGGNS FMPANGLTVA QNQVLNLIKA CPRPEGLNFQ DLKNQLKHMS VS SIKQAVD FLSNEGHIYS TVDDDHFKST DAE

UniProtKB: Replication protein A 32 kDa subunit

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Macromolecule #3: Replication protein A 14 kDa subunit

MacromoleculeName: Replication protein A 14 kDa subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.583714 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MVDMMDLPRS RINAGMLAQF IDKPVCFVGR LEKIHPTGKM FILSDGEGKN GTIELMEPLD EEISGIVEVV GRVTAKATIL CTSYVQFKE DSHPFDLGLY NEAVKIIHDF PQFYPLGIVQ HD

UniProtKB: Replication protein A 14 kDa subunit

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
0.25 mMC9H15O6PTCEP
0.1 mMC32H58N2O8SCHAPSO
2.0 mMMg(CH3COO)2Magnesium acetate
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 21697126
Startup modelType of model: INSILICO MODEL / In silico model: RELION 3D initial model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 1316062
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8rk2:
Human Replication protein A (RPA; trimeric core) - ssDNA complex

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