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- EMDB-19051: RAD51 nucleoprotein filament on double-stranded abasic DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-19051
TitleRAD51 nucleoprotein filament on double-stranded abasic DNA
Map datalocal anisotropy sharpened map
Sample
  • Complex: RAD51 nucleoprotein filament on double-stranded abasic DNA
    • Complex: DNA repair protein
      • Protein or peptide: DNA repair protein RAD51 homolog 1
    • Complex: DNA
      • DNA: 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: 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')
  • Ligand: CALCIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsHomologous recombination / DNA replication / abasic DNA / DNA BINDING PROTEIN
Function / homology
Function and homology information


presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / DNA strand invasion ...presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / DNA strand invasion / mitotic recombination / cellular response to hydroxyurea / DNA strand exchange activity / lateral element / replication-born double-strand break repair via sister chromatid exchange / telomere maintenance via recombination / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / 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) / ATP-dependent DNA damage sensor activity / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / nuclear chromosome / DNA unwinding involved in DNA replication / replication fork processing / Transcriptional Regulation by E2F6 / Presynaptic phase of homologous DNA pairing and strand exchange / ATP-dependent activity, acting on DNA / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / condensed nuclear chromosome / male germ cell nucleus / meiotic cell cycle / cellular response to ionizing radiation / double-strand break repair via homologous recombination / regulation of protein phosphorylation / HDR through Homologous Recombination (HRR) / PML body / Meiotic recombination / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
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
DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAppleby R / Pellegrini L
Funding support United Kingdom, 1 items
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, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_19051.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal anisotropy sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.3 Å/pix.
x 200 pix.
= 260.8 Å		1.3 Å/pix.
x 200 pix.
= 260.8 Å		1.3 Å/pix.
x 200 pix.
= 260.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.304 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.059023358 - 0.089927696
Average (Standard dev.)0.000057128775 (±0.003394697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 260.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : RAD51 nucleoprotein filament on double-stranded abasic DNA

EntireName: RAD51 nucleoprotein filament on double-stranded abasic DNA
Components
  • Complex: RAD51 nucleoprotein filament on double-stranded abasic DNA
    • Complex: DNA repair protein
      • Protein or peptide: DNA repair protein RAD51 homolog 1
    • Complex: DNA
      • DNA: 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: 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')
  • Ligand: CALCIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: RAD51 nucleoprotein filament on double-stranded abasic DNA

SupramoleculeName: RAD51 nucleoprotein filament on double-stranded abasic DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: DNA repair protein

SupramoleculeName: DNA repair protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA repair protein RAD51 homolog 1

MacromoleculeName: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.009125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ...String:
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ERLLAVAERY GLSGSDVLDN VAYARAFNTD HQTQLLYQAS AMMVESRYAL LIVDSATALY RTDYSGRGEL SA RQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQVDGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD

UniProtKB: DNA repair protein RAD51 homolog 1

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Macromolecule #2: DNA (5'-D(P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*TP*GP*(3DR)P*T...

MacromoleculeName: 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')
type: dna / ID: 2
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.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.282902 KDa
SequenceString:
(DT)(DG)(3DR)(DT)(DG)(3DR)(DT)(DG)(3DR)(DT) (DG)(3DR)(DT)(DG)(3DR)(DT)(DG)(3DR) (DT) (DG)(3DR)(DT)(DG)

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Macromolecule #3: DNA (5'-D(P*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP*CP*AP*CP...

MacromoleculeName: 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')
type: dna / ID: 3
Details: The sequence consists of 7 x CAC + CA, as helically averaged version of the DNA sequence: GCTCACGTCTACCACTGCATACC
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.798423 KDa
SequenceString:
(DC)(DA)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DC) (DA)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DC)(DA) (DC)(DC)(DA)

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 16 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 8 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 10084 / Average exposure time: 1.2 sec. / Average electron dose: 52.968 e/Å2
Details: Images were collected in movie mode at 44 frames per movie
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm
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

Final reconstructionApplied symmetry - Helical parameters - Δz: 15.8 Å
Applied symmetry - Helical parameters - Δ&Phi: 56.0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 8853
Segment selectionNumber selected: 1791680 / Software - Name: RELION (ver. 3.1)
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial modelPDB ID:

8br2


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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