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- PDB-6zh8: Cryo-EM structure of DNA-PKcs:DNA -

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Basic information

Entry
Database: PDB / ID: 6zh8
TitleCryo-EM structure of DNA-PKcs:DNA
Components
  • DNA (5'-D(P*AP*CP*TP*AP*AP*AP*AP*A)-3')
  • DNA (5'-D(P*AP*GP*TP*TP*TP*TP*TP*AP*GP*TP*T)-3')
  • DNA-dependent protein kinase catalytic subunit,DNA-PKcs
KeywordsDNA BINDING PROTEIN / Kinase / DNA-PKcs / NHEJ / DNA-repair / DNA-PK
Function / homology
Function and homology information


positive regulation of platelet formation / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex ...positive regulation of platelet formation / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / immunoglobulin V(D)J recombination / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / double-strand break repair via alternative nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / regulation of epithelial cell proliferation / IRF3-mediated induction of type I IFN / telomere capping / U3 snoRNA binding / regulation of hematopoietic stem cell differentiation / maturation of 5.8S rRNA / T cell lineage commitment / negative regulation of cGAS/STING signaling pathway / B cell lineage commitment / positive regulation of double-strand break repair via nonhomologous end joining / ectopic germ cell programmed cell death / somitogenesis / mitotic G1 DNA damage checkpoint signaling / telomere maintenance / activation of innate immune response / small-subunit processome / positive regulation of translation / negative regulation of protein phosphorylation / positive regulation of erythrocyte differentiation / protein-DNA complex / response to gamma radiation / Nonhomologous End-Joining (NHEJ) / brain development / peptidyl-threonine phosphorylation / protein modification process / protein destabilization / regulation of circadian rhythm / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / rhythmic process / intrinsic apoptotic signaling pathway in response to DNA damage / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / T cell differentiation in thymus / heart development / double-stranded DNA binding / peptidyl-serine phosphorylation / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / chromosome, telomeric region / non-specific serine/threonine protein kinase / protein kinase activity / positive regulation of apoptotic process / protein domain specific binding / protein phosphorylation / protein serine kinase activity / innate immune response / protein serine/threonine kinase activity / DNA damage response / chromatin / nucleolus / negative regulation of apoptotic process / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / RNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol
Similarity search - Function
DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, catalytic domain / DNA-dependent protein kinase catalytic subunit, CC5 / DNA-dependent protein kinase catalytic subunit, CC1/2 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC1/2 / NUC194 ...DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, catalytic domain / DNA-dependent protein kinase catalytic subunit, CC5 / DNA-dependent protein kinase catalytic subunit, CC1/2 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC1/2 / NUC194 / PIK-related kinase, FAT / FAT domain / FATC domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-dependent protein kinase catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.14 Å
AuthorsChaplin, A.K. / Hardwick, S.W. / Chirgadze, D.Y. / Blundell, T.L.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z United Kingdom
Citation
Journal: Nat Struct Mol Biol / Year: 2021
Title: Dimers of DNA-PK create a stage for DNA double-strand break repair.
Authors: Amanda K Chaplin / Steven W Hardwick / Shikang Liang / Antonia Kefala Stavridi / Ales Hnizda / Lee R Cooper / Taiana Maia De Oliveira / Dimitri Y Chirgadze / Tom L Blundell /
Abstract: DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent ...DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.
#1: Journal: Nat.Struct.Mol.Biol. / Year: 2020
Title: Dimers of DNA-PK create a stage for DNA-double strand break repair
Authors: Chaplin, A.K. / Hardwick, S.W. / Liang, S. / Stavridi, A.K. / Hnizda, A. / Cooper, L.R. / De Oliveira, T.M. / Chirgadze, D.Y. / Blundell, T.L.
History
DepositionJun 21, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 4, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Jan 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: DNA-dependent protein kinase catalytic subunit,DNA-PKcs
B: DNA (5'-D(P*AP*GP*TP*TP*TP*TP*TP*AP*GP*TP*T)-3')
C: DNA (5'-D(P*AP*CP*TP*AP*AP*AP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)477,8533
Polymers477,8533
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1490 Å2
ΔGint-9 kcal/mol
Surface area165760 Å2
MethodPISA

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Components

#1: Protein DNA-dependent protein kinase catalytic subunit,DNA-PKcs / DNA-PKcs / DNPK1 / p460


Mass: 472056.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA
References: UniProt: P78527, non-specific serine/threonine protein kinase
#2: DNA chain DNA (5'-D(P*AP*GP*TP*TP*TP*TP*TP*AP*GP*TP*T)-3')


Mass: 3369.217 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (5'-D(P*AP*CP*TP*AP*AP*AP*AP*A)-3')


Mass: 2427.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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
1DNA-PKcs bound to DNAORGANELLE OR CELLULAR COMPONENTall0MULTIPLE SOURCES
2DNA-PKcsORGANELLE OR CELLULAR COMPONENT#11NATURAL
3DNAORGANELLE OR CELLULAR COMPONENT#2-#31RECOMBINANT
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.4
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 FIELDBright-field microscopy
Image recordingElectron dose: 52.97 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 350668
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31881 / Symmetry type: POINT

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