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- EMDB-38233: The cryo-EM structure of the RAD51 L1 and L2 loops bound to the l... -

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Basic information

Entry
Database: EMDB / ID: EMD-38233
TitleThe cryo-EM structure of the RAD51 L1 and L2 loops bound to the linker DNA with the blunt end of the nucleosome
Map data
Sample
  • Complex: RAD51-nucleosome complex
    • DNA: DNA (5'-D(P*AP*AP*CP*GP*AP*AP*AP*AP*CP*GP*GP*CP*CP*AP*CP*CP*AP*CP*G)-3')
    • DNA: DNA (5'-D(P*CP*GP*TP*GP*GP*TP*GP*GP*CP*CP*GP*TP*TP*TP*TP*CP*GP*TP*T)-3')
    • Protein or peptide: DNA repair protein RAD51 homolog 1
KeywordsNucleosome / Recombinase / DNA BINDING PROTEIN-DNA Complex
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 / mitotic recombination ...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 / mitotic recombination / DNA strand invasion / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / DNA strand exchange activity / lateral element / telomere maintenance via recombination / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / single-stranded DNA helicase activity / reciprocal meiotic recombination / 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 / Homologous DNA Pairing and Strand Exchange / 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 / meiotic cell cycle / condensed nuclear chromosome / male germ cell nucleus / 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsShioi T / Hatazawa S / Ogasawara M / Takizawa Y / Kurumizaka H
Funding support Japan, 5 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP22K06098 Japan
Japan Society for the Promotion of Science (JSPS)JP23H05475 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121009 Japan
Japan Science and TechnologyJPMJER1901 Japan
Japan Society for the Promotion of Science (JSPS)JP23K14134 Japan
CitationJournal: Nature / Year: 2024
Title: Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.
Authors: Takuro Shioi / Suguru Hatazawa / Eriko Oya / Noriko Hosoya / Wataru Kobayashi / Mitsuo Ogasawara / Takehiko Kobayashi / Yoshimasa Takizawa / Hitoshi Kurumizaka /
Abstract: RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which RAD51 functions at DSB sites in ...RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.
History
DepositionDec 7, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38233.png

Downloads & links

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Map

FileDownload / File: emd_38233.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0043
Minimum - Maximum-0.0053054136 - 0.017786577
Average (Standard dev.)0.00006922742 (±0.00055414706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 296.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_38233_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_38233_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RAD51-nucleosome complex

EntireName: RAD51-nucleosome complex
Components
  • Complex: RAD51-nucleosome complex
    • DNA: DNA (5'-D(P*AP*AP*CP*GP*AP*AP*AP*AP*CP*GP*GP*CP*CP*AP*CP*CP*AP*CP*G)-3')
    • DNA: DNA (5'-D(P*CP*GP*TP*GP*GP*TP*GP*GP*CP*CP*GP*TP*TP*TP*TP*CP*GP*TP*T)-3')
    • Protein or peptide: DNA repair protein RAD51 homolog 1

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Supramolecule #1: RAD51-nucleosome complex

SupramoleculeName: RAD51-nucleosome complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA (5'-D(P*AP*AP*CP*GP*AP*AP*AP*AP*CP*GP*GP*CP*CP*AP*CP*CP*AP*CP...

MacromoleculeName: DNA (5'-D(P*AP*AP*CP*GP*AP*AP*AP*AP*CP*GP*GP*CP*CP*AP*CP*CP*AP*CP*G)-3')
type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 48.595086 KDa
SequenceString: (DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC) ...String:
(DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA) (DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA) (DA)(DA) (DC)(DG)(DC)(DA)(DC)(DG)(DT) (DA)(DC)(DG)(DC)(DG)(DC)(DT)(DG)(DT)(DC) (DC)(DC)(DC) (DC)(DG)(DC)(DG)(DT)(DT) (DT)(DT)(DA)(DA)(DC)(DC)(DG)(DC)(DC)(DA) (DA)(DG)(DG)(DG) (DG)(DA)(DT)(DT)(DA) (DC)(DA)(DC)(DC)(DC)(DA)(DA)(DG)(DA)(DC) (DA)(DC)(DC)(DA)(DG) (DG)(DC)(DA)(DC) (DG)(DA)(DG)(DA)(DC)(DA)(DG)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DC)(DA) (DA)(DC)(DG) (DA)(DA)(DA)(DA)(DC)(DG)(DG)(DC)(DC)(DA) (DC)(DC)(DA)(DC)(DG)

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Macromolecule #2: DNA (5'-D(P*CP*GP*TP*GP*GP*TP*GP*GP*CP*CP*GP*TP*TP*TP*TP*CP*GP*TP...

MacromoleculeName: DNA (5'-D(P*CP*GP*TP*GP*GP*TP*GP*GP*CP*CP*GP*TP*TP*TP*TP*CP*GP*TP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 48.952062 KDa
SequenceString: (DC)(DG)(DT)(DG)(DG)(DT)(DG)(DG)(DC)(DC) (DG)(DT)(DT)(DT)(DT)(DC)(DG)(DT)(DT)(DG) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DC)(DT) (DG)(DT)(DC)(DT)(DC)(DG)(DT)(DG)(DC)(DC) (DT) (DG)(DG)(DT)(DG)(DT)(DC) ...String:
(DC)(DG)(DT)(DG)(DG)(DT)(DG)(DG)(DC)(DC) (DG)(DT)(DT)(DT)(DT)(DC)(DG)(DT)(DT)(DG) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DC)(DT) (DG)(DT)(DC)(DT)(DC)(DG)(DT)(DG)(DC)(DC) (DT) (DG)(DG)(DT)(DG)(DT)(DC)(DT)(DT) (DG)(DG)(DG)(DT)(DG)(DT)(DA)(DA)(DT)(DC) (DC)(DC) (DC)(DT)(DT)(DG)(DG)(DC)(DG) (DG)(DT)(DT)(DA)(DA)(DA)(DA)(DC)(DG)(DC) (DG)(DG)(DG) (DG)(DG)(DA)(DC)(DA)(DG) (DC)(DG)(DC)(DG)(DT)(DA)(DC)(DG)(DT)(DG) (DC)(DG)(DT)(DT) (DT)(DA)(DA)(DG)(DC) (DG)(DG)(DT)(DG)(DC)(DT)(DA)(DG)(DA)(DG) (DC)(DT)(DG)(DT)(DC) (DT)(DA)(DC)(DG) (DA)(DC)(DC)(DA)(DA)(DT)(DT)(DG)(DA)(DG) (DC)(DG)(DG)(DC)(DC)(DT) (DC)(DG)(DG) (DC)(DA)(DC)(DC)(DG)(DG)(DG)(DA)(DT)(DT) (DC)(DT)(DG)(DA)(DT)

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Macromolecule #3: DNA repair protein RAD51 homolog 1

MacromoleculeName: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.291398 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMAMQMQL EANADTSVEE ESFGPQPISR LEQCGINAND VKKLEEAGFH TVEAVAYAPK KELINIKGIS EAKADKILAE AAKLVPMGF TTATEFHQRR SEIIQITTGS KELDKLLQGG IETGSITEMF GEFRTGKTQI CHTLAVTCQL PIDRGGGEGK A MYIDTEGT ...String:
GSHMAMQMQL EANADTSVEE ESFGPQPISR LEQCGINAND VKKLEEAGFH TVEAVAYAPK KELINIKGIS EAKADKILAE AAKLVPMGF TTATEFHQRR SEIIQITTGS KELDKLLQGG IETGSITEMF GEFRTGKTQI CHTLAVTCQL PIDRGGGEGK A MYIDTEGT FRPERLLAVA ERYGLSGSDV LDNVAYARAF NTDHQTQLLY QASAMMVESR YALLIVDSAT ALYRTDYSGR GE LSARQMH LARFLRMLLR LADEFGVAVV ITNQVVAQVD GAAMFAADPK KPIGGNIIAH ASTTRLYLRK GRGETRICKI YDS PCLPEA EAMFAINADG VGDAKD

UniProtKB: DNA repair protein RAD51 homolog 1

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5nwl


Details: 5Y0C and 7OHC were also used.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 10869
FSC plot (resolution estimation)

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