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- PDB-8rcf: RAD51 nucleoprotein filament on double-stranded abasic DNA -

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Basic information

Entry
Database: PDB / ID: 8rcf
TitleRAD51 nucleoprotein filament on double-stranded abasic DNA
Components
  • DNA (5'-D(P*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*A)-3')
  • DNA (5'-D(P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*G)-3')
  • DNA repair protein RAD51 homolog 1
KeywordsDNA BINDING PROTEIN / Homologous recombination / DNA replication / abasic DNA
Function / homology
Function and homology information


presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to cisplatin ...presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to cisplatin / DNA strand invasion / cellular response to hydroxyurea / mitotic recombination / DNA strand exchange activity / replication-born double-strand break repair via sister chromatid exchange / lateral element / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination / single-stranded DNA helicase activity / reciprocal meiotic recombination / Homologous DNA Pairing and Strand Exchange / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / HDR through Single Strand Annealing (SSA) / regulation of double-strand break repair via homologous recombination / ATP-dependent DNA damage sensor activity / nuclear chromosome / Impaired BRCA2 binding to RAD51 / Transcriptional Regulation by E2F6 / replication fork processing / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / interstrand cross-link repair / condensed chromosome / DNA polymerase binding / condensed nuclear chromosome / cellular response to ionizing radiation / meiotic cell cycle / male germ cell nucleus / cellular response to gamma radiation / double-strand break repair via homologous recombination / PML body / HDR through Homologous Recombination (HRR) / response to toxic substance / Meiotic recombination / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / response to xenobiotic stimulus / DNA repair / DNA damage response / centrosome / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / protein-containing complex / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain ...DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAppleby, R. / Pellegrini, L.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust221892/Z/20/Z United Kingdom
CitationJournal: Mol Cell / Year: 2024
Title: RAD51 protects abasic sites to prevent replication fork breakage.
Authors: Yodhara Wijesekara Hanthi / Miguel Angel Ramirez-Otero / Robert Appleby / Anna De Antoni / Luay Joudeh / Vincenzo Sannino / Salli Waked / Alessandra Ardizzoia / Viviana Barra / Daniele ...Authors: Yodhara Wijesekara Hanthi / Miguel Angel Ramirez-Otero / Robert Appleby / Anna De Antoni / Luay Joudeh / Vincenzo Sannino / Salli Waked / Alessandra Ardizzoia / Viviana Barra / Daniele Fachinetti / Luca Pellegrini / Vincenzo Costanzo /
Abstract: Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture ...Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.
History
DepositionDec 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
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Revision 2.0May 21, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
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Item: _atom_site.auth_asym_id / _atom_site.auth_seq_id ..._atom_site.auth_asym_id / _atom_site.auth_seq_id / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_nonpoly_scheme.pdb_strand_id / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / Group: Experimental summary / Data content type: EM metadata / Category: em_admin / Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA repair protein RAD51 homolog 1
B: DNA repair protein RAD51 homolog 1
C: DNA repair protein RAD51 homolog 1
D: DNA repair protein RAD51 homolog 1
E: DNA repair protein RAD51 homolog 1
F: DNA repair protein RAD51 homolog 1
G: DNA repair protein RAD51 homolog 1
H: DNA repair protein RAD51 homolog 1
I: DNA (5'-D(P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*G)-3')
J: DNA (5'-D(P*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)313,85334
Polymers309,15410
Non-polymers4,69924
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area52050 Å2
ΔGint-381 kcal/mol
Surface area97320 Å2
MethodPISA

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Components

#1: Protein
DNA repair protein RAD51 homolog 1 / HsRAD51 / hRAD51 / RAD51 homolog A


Mass: 37009.125 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51, RAD51A, RECA / Production host: Escherichia coli (E. coli) / References: UniProt: Q06609
#2: DNA chain DNA (5'-D(P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*G)-3')


Mass: 6282.902 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: 3DR refers to an abasic nucleotide. The ribose of the abasic nucleotide is a tetrahydrofurane lacking the hydroxyl group that would naturally be present on position C1 after base hydrolysis. ...Details: 3DR refers to an abasic nucleotide. The ribose of the abasic nucleotide is a tetrahydrofurane lacking the hydroxyl group that would naturally be present on position C1 after base hydrolysis. The sequence consists of 7 x TG(3DR) + TG, as helically averaged version of the DNA sequence: GGTAT(3DR)CA(3DR)TG(3DR)TA(3DR)AC(3DR)TGAGC, which contains 5 central, equally spaced abasic sites flanked by 5 nucleotides.
Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (5'-D(P*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*A)-3')


Mass: 6798.423 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The sequence consists of 7 x CAC + CA, as helically averaged version of the DNA sequence: GCTCACGTCTACCACTGCATACC
Source: (synth.) Homo sapiens (human)
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Ca
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1RAD51 nucleoprotein filament on double-stranded abasic DNACOMPLEX#1-#30MULTIPLE SOURCES
2DNA repair proteinCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 / Calibrated magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 52.968 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 10084
Details: Images were collected in movie mode at 44 frames per movie

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Coot0.9.8.1model fitting
12RELION3.13D reconstruction
13PHENIX1.21rc1-4985model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 56 ° / Axial rise/subunit: 15.8 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1791680
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8853 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 8BR2

8br2


Accession code: 8BR2 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00820573
ELECTRON MICROSCOPYf_angle_d0.62227974
ELECTRON MICROSCOPYf_dihedral_angle_d13.2187749
ELECTRON MICROSCOPYf_chiral_restr0.0443186
ELECTRON MICROSCOPYf_plane_restr0.0053447

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