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- PDB-8jnf: The cryo-EM structure of the RAD51 filament bound to the nucleosome -

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Basic information

Entry
Database: PDB / ID: 8jnf
TitleThe cryo-EM structure of the RAD51 filament bound to the nucleosome
Components
  • DNA (153-MER)
  • DNA (156-MER)
  • DNA repair protein RAD51 homolog 1
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1
  • Histone H4
KeywordsDNA BINDING PROTEIN/DNA / Nucleosome / Recombinase / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / cellular response to cisplatin / nuclear ubiquitin ligase complex / DNA strand invasion ...presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / cellular response to cisplatin / nuclear ubiquitin ligase complex / DNA strand invasion / mitotic recombination / cellular response to hydroxyurea / cellular response to camptothecin / replication-born double-strand break repair via sister chromatid exchange / lateral element / DNA strand exchange activity / Impaired BRCA2 binding to PALB2 / regulation of DNA damage checkpoint / telomere maintenance via recombination / reciprocal meiotic recombination / single-stranded DNA helicase activity / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / HDR through Single Strand Annealing (SSA) / 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) / nuclear chromosome / Resolution of D-loop Structures through Holliday Junction Intermediates / Transcriptional Regulation by E2F6 / Impaired BRCA2 binding to RAD51 / replication fork processing / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / negative regulation of tumor necrosis factor-mediated signaling pathway / ATP-dependent activity, acting on DNA / interstrand cross-link repair / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / condensed chromosome / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / DNA polymerase binding / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / epigenetic regulation of gene expression / Deposition of new CENPA-containing nucleosomes at the centromere / telomere organization / Interleukin-7 signaling / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / RNA Polymerase I Promoter Opening / Inhibition of DNA recombination at telomere / Assembly of the ORC complex at the origin of replication / Meiotic synapsis / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / DNA methylation / Condensation of Prophase Chromosomes / condensed nuclear chromosome / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / male germ cell nucleus / cellular response to ionizing radiation / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / meiotic cell cycle / Regulation of endogenous retroelements by KRAB-ZFP proteins / innate immune response in mucosa / Defective pyroptosis / HDACs deacetylate histones / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / lipopolysaccharide binding / Transcriptional regulation by small RNAs / HDMs demethylate histones / Formation of the beta-catenin:TCF transactivating complex / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / cellular response to gamma radiation / protein-DNA complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / PML body / NoRC negatively regulates rRNA expression / Negative Regulation of CDH1 Gene Transcription / G2/M DNA damage checkpoint / PKMTs methylate histone lysines / B-WICH complex positively regulates rRNA expression / double-strand break repair via homologous recombination / DNA Damage/Telomere Stress Induced Senescence / Meiotic recombination / Pre-NOTCH Transcription and Translation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / HDR through Homologous Recombination (HRR) / Activation of anterior HOX genes in hindbrain development during early embryogenesis
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 / SAM-like Helix-hairpin-helix tandem ...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 / SAM-like Helix-hairpin-helix tandem / : / Histone H2A conserved site / Histone H2A signature. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1 / DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.91 Å
AuthorsShioi, T. / Hatazawa, S. / Ogasawara, M. / Takizawa, Y. / Kurumizaka, H.
Funding support Japan, 5items
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
DepositionJun 6, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Apr 17, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Apr 24, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.4May 8, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.1
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.1
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: DNA (156-MER)
J: DNA (153-MER)
K: DNA repair protein RAD51 homolog 1
L: DNA repair protein RAD51 homolog 1
M: DNA repair protein RAD51 homolog 1
N: DNA repair protein RAD51 homolog 1
O: DNA repair protein RAD51 homolog 1
P: DNA repair protein RAD51 homolog 1


Theoretical massNumber of molelcules
Total (without water)431,21916
Polymers431,21916
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 14 molecules AEBFCGDHKLMNOP

#1: Protein Histone H3.1 / Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone ...Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone H3/i / Histone H3/j / Histone H3/k / Histone H3/l


Mass: 15719.445 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H3C1, H3FA, HIST1H3A, H3C2, H3FL, HIST1H3B, H3C3, H3FC HIST1H3C, H3C4, H3FB, HIST1H3D, H3C6, H3FD, HIST1H3E, H3C7, H3FI, HIST1H3F, H3C8, H3FH, HIST1H3G, H3C10, H3FK, HIST1H3H, H3C11, H3FF, ...Gene: H3C1, H3FA, HIST1H3A, H3C2, H3FL, HIST1H3B, H3C3, H3FC HIST1H3C, H3C4, H3FB, HIST1H3D, H3C6, H3FD, HIST1H3E, H3C7, H3FI, HIST1H3F, H3C8, H3FH, HIST1H3G, H3C10, H3FK, HIST1H3H, H3C11, H3FF, HIST1H3I, H3C12, H3FJ, HIST1H3J
Production host: Escherichia coli (E. coli) / References: UniProt: P68431
#2: Protein Histone H4


Mass: 11676.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H4C1 / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14447.825 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE / Production host: Escherichia coli (E. coli) / References: UniProt: P04908
#4: Protein Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 14217.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC11, H2BFR, HIST1H2BJ / Production host: Escherichia coli (E. coli) / References: UniProt: P06899
#7: Protein
DNA repair protein RAD51 homolog 1 / HsRAD51 / hRAD51 / RAD51 homolog A


Mass: 37291.398 Da / Num. of mol.: 6
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

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (156-MER)


Mass: 47976.699 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#6: DNA chain DNA (153-MER)


Mass: 47371.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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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: RAD51-nucleosome complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60.52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53864 / Symmetry type: POINT

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