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- PDB-6r8y: Cryo-EM structure of NCP-6-4PP(-1)-UV-DDB -

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Basic information

Entry
Database: PDB / ID: 6r8y
TitleCryo-EM structure of NCP-6-4PP(-1)-UV-DDB
Components
  • (DNA damage-binding protein ...) x 2
  • (Histone H4) x 2
  • (Human alpha-satellite DNA (145- ...) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1Histone H3
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 / UV-damage excision repair / histone H2A monoubiquitination / Cul4A-RING E3 ubiquitin ligase complex / WD40-repeat domain binding / biological process involved in interaction with symbiont / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion ...positive regulation by virus of viral protein levels in host cell / : / Cul4B-RING E3 ubiquitin ligase complex / UV-damage excision repair / histone H2A monoubiquitination / Cul4A-RING E3 ubiquitin ligase complex / WD40-repeat domain binding / biological process involved in interaction with symbiont / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion / Cul4-RING E3 ubiquitin ligase complex / cullin family protein binding / negative regulation of megakaryocyte differentiation / Packaging Of Telomere Ends / positive regulation of gluconeogenesis / negative regulation of tumor necrosis factor-mediated signaling pathway / regulation of mitotic cell cycle phase transition / Chromatin modifying enzymes / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / proteasomal protein catabolic process / nucleotide-excision repair, DNA damage recognition / DNA replication-independent chromatin assembly / global genome nucleotide-excision repair / nucleotide-excision repair, DNA duplex unwinding / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / nucleotide-excision repair, DNA incision, 5'-to lesion / protein autoubiquitination / Meiotic synapsis / positive regulation of viral genome replication / nucleotide-excision repair, preincision complex assembly / heterochromatin assembly => GO:0031507 / pyrimidine dimer repair / telomere organization / RNA Polymerase I Promoter Opening / SUMOylation of chromatin organization proteins / response to UV / Interleukin-7 signaling / DNA replication-dependent chromatin assembly / Defective pyroptosis / nucleotide-excision repair, DNA incision / DNA methylation / HCMV Late Events / innate immune response in mucosa / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / : / Condensation of Prophase Chromosomes / PRC2 methylates histones and DNA / : / RNA Polymerase I Promoter Escape / HDACs deacetylate histones / nucleotide-excision repair / nuclear chromosome / TP53 Regulates Transcription of DNA Repair Genes / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Recognition of DNA damage by PCNA-containing replication complex / NoRC negatively regulates rRNA expression / Formation of the beta-catenin:TCF transactivating complex / DNA Damage Recognition in GG-NER / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / B-WICH complex positively regulates rRNA expression / transcription-coupled nucleotide-excision repair / DNA-templated transcription, initiation / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Metalloprotease DUBs / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / Dual Incision in GG-NER / DNA Damage/Telomere Stress Induced Senescence / G2/M DNA damage checkpoint / lipopolysaccharide binding / nucleosome assembly / RMTs methylate histone arginines / positive regulation of protein catabolic process / HCMV Early Events / Formation of Incision Complex in GG-NER / Pre-NOTCH Transcription and Translation / HDMs demethylate histones / Dual incision in TC-NER / PKMTs methylate histone lysines / Gap-filling DNA repair synthesis and ligation in TC-NER / Meiotic recombination / protein-macromolecule adaptor activity / nucleosome / Activation of anterior HOX genes in hindbrain development during early embryogenesis / post-translational protein modification / UCH proteinases / Transcriptional regulation of granulopoiesis / site of double-strand break / Neddylation / rhythmic process / E3 ubiquitin ligases ubiquitinate target proteins / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / proteasome-mediated ubiquitin-dependent protein catabolic process
Similarity search - Function
DNA damage-binding protein 2 / DNA damage-binding protein 1 / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A signature. ...DNA damage-binding protein 2 / DNA damage-binding protein 1 / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A signature. / Histone H2A conserved site / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / Histone H4 signature. / Histone H4, conserved site / Histone H4 / Histone H4 / Centromere kinetochore component CENP-T histone fold / CENP-T/Histone H4, histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF) / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Core histone H2A/H2B/H3/H4 / Histone H2A/H2B/H3 / Histone-fold / : / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
DNA damage-binding protein 1 / Histone H4 / Histone H3.1 / DNA (> 100) / Histone H2B type 1-J / Histone H2A type 1-B/E / DNA (> 10) / DNA / DNA damage-binding protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 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.
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
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: Human alpha-satellite DNA (145-MER)
J: Human alpha-satellite DNA (145-MER) with a 6-4PP at positions 95-96
K: DNA damage-binding protein 1
L: DNA damage-binding protein 2


Theoretical massNumber of molelcules
Total (without water)377,01812
Polymers377,01812
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66590 Å2
ΔGint-420 kcal/mol
Surface area124050 Å2
MethodPISA

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Components

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Protein , 5 types, 8 molecules AEBCGDHF

#1: 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
#2: Protein Histone H4 /


Mass: 11676.703 Da / Num. of mol.: 1
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
Production host: Escherichia coli (E. coli) / Strain (production host): JM109(DE3) / 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: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Production host: Escherichia coli BL21(DE3) (bacteria) / 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: HIST1H2BJ, H2BFR / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P06899
#5: Protein Histone H4 /


Mass: 11394.426 Da / Num. of mol.: 1
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
Production host: Escherichia coli (E. coli) / Strain (production host): JM109(DE3) / References: UniProt: P62805

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

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


Mass: 44780.672 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#7: DNA chain Human alpha-satellite DNA (145-MER) with a 6-4PP at positions 95-96


Mass: 44709.645 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: 145 bp human alpha-satellite with a 6-4 pyrimidine-pyrimidone (6-4PP) at base positions 95-96
Source: (synth.) Homo sapiens (human)

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

#8: Protein 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: 127425.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16531
#9: 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: 48261.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92466

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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 6-4PP containing nucleosomeCOMPLEX#1-#90MULTIPLE SOURCES
2histonesHistoneCOMPLEX#1, #3-#41RECOMBINANT
3DNA damage-binding proteinsCOMPLEX#8-#91RECOMBINANT
4DNACOMPLEX#6-#71RECOMBINANT
5histonesHistoneCOMPLEX#2, #51RECOMBINANT
Molecular weightValue: 0.199 MDa / Experimental value: NO
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
22Escherichia coli BL21(DE3) (bacteria)469008
33Trichoplusia ni (cabbage looper)7111
44synthetic construct (others)32630
55Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium chlorideNaClSodium chloride1
250 mMHEPES1
30.25 mMTCEP1
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 / Nominal magnification: 130000 X / Cs: 0 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: dev_3318: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4GctfCTF correction
7Coot0.8.9.1model fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION23D reconstruction
19REFMAC5.8.0238model refinementDNA ONLY
20PHENIXdev-3318model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
14ZUXI1
24ZUXJ1
35Y0CA1
45Y0CB1
55Y0CC1
65Y0CD1
75Y0CE1
85Y0CF1
95Y0CG1
105Y0CH1
114.0E+54B1
123EI4A1

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