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- PDB-6r92: Cryo-EM structure of NCP-THF2(+1)-UV-DDB class B -

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Basic information

Entry
Database: PDB / ID: 6r92
TitleCryo-EM structure of NCP-THF2(+1)-UV-DDB class B
Components
  • (DNA damage-binding protein ...) x 2
  • (Human alpha-satellite DNA (145- ...) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1Histone H3
  • Histone H4
KeywordsDNA BINDING PROTEIN / DNA damage / Nucleosome / 6-4 photoproduct
Function / homology
Function and homology information


positive regulation by virus of viral protein levels in host cell / Cul4B-RING E3 ubiquitin ligase complex / positive regulation of viral release from host cell / Cul4A-RING E3 ubiquitin ligase complex / UV-damage excision repair / WD40-repeat domain binding / histone H2A monoubiquitination / cullin-RING ubiquitin ligase complex / interaction with symbiont / Cul4-RING E3 ubiquitin ligase complex ...positive regulation by virus of viral protein levels in host cell / Cul4B-RING E3 ubiquitin ligase complex / positive regulation of viral release from host cell / Cul4A-RING E3 ubiquitin ligase complex / UV-damage excision repair / WD40-repeat domain binding / histone H2A monoubiquitination / cullin-RING ubiquitin ligase complex / interaction with symbiont / Cul4-RING E3 ubiquitin ligase complex / cullin family protein binding / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion / negative regulation of megakaryocyte differentiation / positive regulation of gluconeogenesis / CENP-A containing nucleosome assembly / negative regulation of tumor necrosis factor-mediated signaling pathway / DNA replication-independent nucleosome assembly / telomere capping / positive regulation of viral genome replication / interleukin-7-mediated signaling pathway / protein autoubiquitination / chromatin silencing / regulation of mitotic cell cycle phase transition / response to UV / pyrimidine dimer repair / telomere organization / DNA replication-dependent nucleosome assembly / innate immune response in mucosa / nuclear nucleosome / rDNA heterochromatin assembly / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / regulation of gene silencing / nucleotide-excision repair, DNA damage recognition / nuclear chromosome / DNA-templated transcription, initiation / nucleotide-excision repair, DNA duplex unwinding / proteasomal protein catabolic process / global genome nucleotide-excision repair / nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / regulation of megakaryocyte differentiation / nucleotide-excision repair, DNA incision, 5'-to lesion / DNA damage response, detection of DNA damage / nucleotide-excision repair, DNA incision / positive regulation of protein catabolic process / nucleosome assembly / protein-macromolecule adaptor activity / regulation of circadian rhythm / lipopolysaccharide binding / nucleosome / transcription-coupled nucleotide-excision repair / rhythmic process / cell junction / proteasome-mediated ubiquitin-dependent protein catabolic process / double-strand break repair via nonhomologous end joining / post-translational protein modification / chromatin organization / protein polyubiquitination / nuclear chromosome, telomeric region / ubiquitin-dependent protein catabolic process / Wnt signaling pathway / killing of cells of other organism / antibacterial humoral response / damaged DNA binding / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / protein ubiquitination / protein deubiquitination / cadherin binding / defense response to Gram-negative bacterium / protein heterodimerization activity / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / nuclear chromatin / DNA repair / protein domain specific binding / cellular response to DNA damage stimulus / protein-containing complex binding / cellular protein metabolic process / viral process / negative regulation of apoptotic process / host cell nucleus / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / extracellular region / nucleoplasm / nucleus / cytosol / cytoplasm
WD domain, G-beta repeat / Histone H4, conserved site / Histone H3/CENP-A / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Histone H2A/H2B/H3 / Histone-fold ...WD domain, G-beta repeat / Histone H4, conserved site / Histone H3/CENP-A / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Histone H2A/H2B/H3 / Histone-fold / WD40/YVTN repeat-like-containing domain superfamily / WD40-repeat-containing domain / WD40 repeat, conserved site / WD40 repeat / DNA damage-binding protein 1 / Histone H2A conserved site / DNA damage-binding protein 2 / CENP-T/Histone H4, histone fold / Histone H2A, C-terminal domain / WD40-repeat-containing domain superfamily / Core histone H2A/H2B/H3/H4 / CPSF A subunit region / Centromere kinetochore component CENP-T histone fold / C-terminus of histone H2A
Histone H2B type 1-J / Histone H2A type 1-B/E / Histone H3.1 / DNA damage-binding protein 1 / DNA damage-binding protein 2 / Histone H4
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsMatsumoto, S. / Cavadini, S. / Bunker, R.D. / Thoma, N.H.
Funding support Switzerland, Japan, 7items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_160734/1 Switzerland
European Union666068
European Commission667951
European Commission705354
Japan Society for the Promotion of ScienceJP18H05534 Japan
Japan Society for the Promotion of ScienceJP16H06307 Japan
Japan Agency for Medical Research and Development (AMED)JP18am0101076 Japan
CitationJournal: Nature / Year: 2019
Title: DNA damage detection in nucleosomes involves DNA register shifting.
Authors: Syota Matsumoto / Simone Cavadini / Richard D Bunker / Ralph S Grand / Alessandro Potenza / Julius Rabl / Junpei Yamamoto / Andreas D Schenk / Dirk Schübeler / Shigenori Iwai / Kaoru ...Authors: Syota Matsumoto / Simone Cavadini / Richard D Bunker / Ralph S Grand / Alessandro Potenza / Julius Rabl / Junpei Yamamoto / Andreas D Schenk / Dirk Schübeler / Shigenori Iwai / Kaoru Sugasawa / Hitoshi Kurumizaka / Nicolas H Thomä /
Abstract: Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced ...Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Author supporting evidence / Data collection / Database references
Category: citation / em_image_scans / em_single_particle_entity
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
I: Human alpha-satellite DNA (145-MER)
J: Human alpha-satellite DNA (145-MER) with abasic sites at positions 93-94
L: DNA damage-binding protein 2
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
K: DNA damage-binding protein 1,DNA damage-binding protein 1


Theoretical massNumber of molelcules
Total (without water)381,70012
Polymers381,70012
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area64830 Å2
ΔGint-418 kcal/mol
Surface area122820 Å2
MethodPISA

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Components

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Human alpha-satellite DNA (145- ... , 2 types, 2 molecules IJ

#1: DNA chain Human alpha-satellite DNA (145-MER)


Mass: 44756.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: DNA chain Human alpha-satellite DNA (145-MER) with abasic sites at positions 93-94


Mass: 44452.434 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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DNA damage-binding protein ... , 2 types, 2 molecules LK

#3: Protein DNA damage-binding protein 2 / DDB p48 subunit / DDBb / Damage-specific DNA-binding protein 2 / UV-damaged DNA-binding protein 2 / UV-DDB 2


Mass: 50601.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92466
#8: Protein DNA damage-binding protein 1,DNA damage-binding protein 1 / DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / ...DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / HBV X-associated protein 1 / XAP-1 / UV-damaged DNA-binding factor / UV-damaged DNA-binding protein 1 / UV-DDB 1 / XPE-binding factor / XPE-BF / Xeroderma pigmentosum group E-complementing protein / XPCe


Mass: 129766.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Cell line (production host): High five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16531

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Protein , 4 types, 8 molecules AEBFCGDH

#4: Protein Histone H3.1 / Histone H3 / 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: HIST1H3A, H3FA, HIST1H3B, H3FL, HIST1H3C, H3FC, HIST1H3D, H3FB, HIST1H3E, H3FD, HIST1H3F, H3FI, HIST1H3G, H3FH, HIST1H3H, H3FK, HIST1H3I, H3FF, HIST1H3J, H3FJ
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P68431
#5: 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: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, ...Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Cell line (production host): JM109(DE3) / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#6: 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: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04908
#7: 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: HIST1H2BJ, H2BFR / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P06899

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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

Component
IDNameTypeEntity IDParent-IDSource
1UV-DDB bound to a THF2 (+1) containing nucleosome class BCOMPLEX#1-#80MULTIPLE SOURCES
2DNACOMPLEX#1-#21RECOMBINANT
3DNA damage-binding protein 1(2), DNA damage-binding protein 2COMPLEX#3, #81RECOMBINANT
4Histone H3.1, Histone H2A, Histone H2BHistone H3COMPLEX#4, #6-#71RECOMBINANT
5Histone H4COMPLEX#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
55Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32630
33Trichoplusia ni (cabbage looper)7111
44Escherichia coli BL21(DE3) (bacteria)469008
55Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_3318: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4GctfCTF correction
7Coot0.8.9.1model fitting
12RELION23D reconstruction
19REFMAC5.8.0238model refinementDNA only
20PHENIXdev-3318model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48925 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-ID
14ZUX

4zux

I1
24ZUX

4zux

J1
35Y0C

5y0c

A1
45Y0C

5y0c

B1
55Y0C

5y0c

C1
65Y0C

5y0c

D1
75Y0C

5y0c

E1
85Y0C

5y0c

F1
95Y0C

5y0c

G1
105Y0C

5y0c

H1
114.0E+54B1
123EI4

3ei4

A1

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