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- PDB-6r90: Cryo-EM structure of NCP-THF2(+1)-UV-DDB class A -

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Entry
Database: PDB / ID: 6r90
TitleCryo-EM structure of NCP-THF2(+1)-UV-DDB class A
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 H3.1
  • Histone H4
KeywordsDNA BINDING PROTEIN / DNA damage / Nucleosome / 6-4 photoproduct
Function / homology
Function and homology information


Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Recognition of DNA damage by PCNA-containing replication complex / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Meiotic synapsis / Interleukin-7 signaling / Packaging Of Telomere Ends / Pre-NOTCH Transcription and Translation / Formation of the beta-catenin:TCF transactivating complex ...Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Recognition of DNA damage by PCNA-containing replication complex / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Meiotic synapsis / Interleukin-7 signaling / Packaging Of Telomere Ends / Pre-NOTCH Transcription and Translation / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence / Condensation of Prophase Chromosomes / Senescence-Associated Secretory Phenotype (SASP) / RNA Polymerase I Promoter Opening / Dual Incision in GG-NER / Deposition of new CENPA-containing nucleosomes at the centromere / Formation of TC-NER Pre-Incision Complex / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / TP53 Regulates Transcription of DNA Repair Genes / G2/M DNA damage checkpoint / RNA Polymerase I Promoter Escape / DNA Damage Recognition in GG-NER / E3 ubiquitin ligases ubiquitinate target proteins / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Neddylation / Estrogen-dependent gene expression / Meiotic recombination / Amyloid fiber formation / Factors involved in megakaryocyte development and platelet production / Formation of Incision Complex in GG-NER / Processing of DNA double-strand break ends / DNA Damage/Telomere Stress Induced Senescence / NoRC negatively regulates rRNA expression / HDACs deacetylate histones / PKMTs methylate histone lysines / HDMs demethylate histones / HATs acetylate histones / RMTs methylate histone arginines / Chromatin modifying enzymes / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SUMOylation of chromatin organization proteins / Nonhomologous End-Joining (NHEJ) / B-WICH complex positively regulates rRNA expression / DNA methylation / Transcriptional regulation by small RNAs / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / UCH proteinases / Ub-specific processing proteases / Metalloprotease DUBs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / positive regulation by virus of viral protein levels in host cell / Cul4B-RING E3 ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / UV-damage excision repair / positive regulation of viral release from host cell / histone H2A monoubiquitination / WD40-repeat domain binding / positive regulation of gluconeogenesis / interaction with symbiont / response to UV-B / 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 / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / interleukin-7-mediated signaling pathway / positive regulation of viral genome replication / regulation of mitotic cell cycle phase transition / protein autoubiquitination / DNA replication-dependent nucleosome assembly / telomere organization / innate immune response in mucosa / response to UV / nuclear nucleosome / pyrimidine dimer repair / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / nucleosomal DNA binding / DNA-templated transcription, initiation / regulation of gene silencing by miRNA / nuclear chromosome / nucleotide-excision repair, DNA damage recognition / nucleotide-excision repair, DNA duplex unwinding / regulation of gene silencing / global genome nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / proteasomal protein catabolic process / regulation of megakaryocyte differentiation / nucleotide-excision repair / nucleotide-excision repair, DNA incision, 5'-to lesion
Mono-functional DNA-alkylating methyl methanesulfonate N-term / Histone H4 signature. / Histone H3 signature 1. / Histone H2B signature. / Trp-Asp (WD) repeats signature. / Histone H3 signature 2. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / C-terminus of histone H2A / Histone H2A signature. ...Mono-functional DNA-alkylating methyl methanesulfonate N-term / Histone H4 signature. / Histone H3 signature 1. / Histone H2B signature. / Trp-Asp (WD) repeats signature. / Histone H3 signature 2. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / C-terminus of histone H2A / Histone H2A signature. / Centromere kinetochore component CENP-T histone fold / WD40 repeat, conserved site / 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 / Histone H4, conserved site / CPSF A subunit region / DNA damage-binding protein 1 / Histone H2A, C-terminal domain / Histone H2A conserved site / DNA damage-binding protein 2 / CENP-T/Histone H4, histone fold / WD40-repeat-containing domain superfamily / Core histone H2A/H2B/H3/H4 / WD domain, G-beta repeat / Histone H2B / WD40 repeat / Histone H3/CENP-A
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1 / DNA damage-binding protein 1 / DNA damage-binding protein 2
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsMatsumoto, S. / Cavadini, S. / Bunker, R.D. / Thoma, N.H.
Funding supportSwitzerland , Japan , 7件
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_160734/1Switzerland
European Union666068
European Commission667951
European Commission705354
Japan Society for the Promotion of ScienceJP18H05534Japan
Japan Society for the Promotion of ScienceJP16H06307Japan
Japan Agency for Medical Research and Development (AMED)JP18am0101076Japan
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 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
DateDeposition: Apr 2, 2019 / Release: Jun 12, 2019
RevisionDateData content typeProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release

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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 abasic sites at positions 93-94
K: DNA damage-binding protein 1
L: DNA damage-binding protein 2


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


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area63680 Å2
ΔGint-433 kcal/mol
Surface area121970 Å2
MethodPISA

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Components

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Protein/peptide , 4 types, 8 molecules AEBFCGDH

#1: Protein/peptide Histone H3.1 / 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/peptide 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, 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
#3: Protein/peptide 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/peptide 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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Human alpha-satellite DNA (145- ... , 2 types, 2 molecules IJ

#5: 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)
#6: 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 KL

#7: Protein/peptide DNA damage-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
#8: Protein/peptide 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

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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

Type: COMPLEX

IDNameEntity IDParent-IDSource
1UV-DDB bound to a THF2 containing nucleosome class A1, 2, 3, 4, 5, 6, 7, 80MULTIPLE SOURCES
2Histone H3.1, Histone H2A, Histone H2B1,3,41RECOMBINANT
3Histone H421RECOMBINANT
4DNA5,61RECOMBINANT
5DNA damage-binding protein 1(2), DNA damage-binding protein 27,81RECOMBINANT
Molecular weightValue: 0.199 MDa / Experimental value: NO
Source (natural)

Ncbi tax-ID: 9606 / Organism: Homo sapiens (human)

IDEntity assembly-ID
22
33
44
55
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Escherichia coli (E. coli)562
44synthetic construct (others)32630
55Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.4
Buffer component

Buffer-ID: 1

IDConc.NameFormula
150 mMSodium chlorideNaClSodium chloride
250 mMHEPES
30.25 mMTCEP
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 µns / 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: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128763 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
14ZUXI
24ZUXJ
35Y0CA
45Y0CB
55Y0CC
65Y0CD
75Y0CE
85Y0CF
95Y0CG
105Y0CH
114.0E+54B
123EI4A

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