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.
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: Protein
X-rayrepaircross-complementingprotein5 / 86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase ...86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase II 80 kDa subunit / CTC box-binding factor 85 kDa subunit / CTCBF / DNA repair protein XRCC5 / Ku80 / Ku86 / Lupus Ku autoantigen protein p86 / Nuclear factor IV / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining)
Mass: 21454.877 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2 / Production host: Escherichia coli (E. coli) References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
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Experimental details
-
Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
-
Sample preparation
Component
ID
Name
Type
Entity ID
Parent-ID
Source
1
DNA-PKcs in complex with Ku80 C-terminal domain
ORGANELLEORCELLULARCOMPONENT
all
0
MULTIPLESOURCES
2
DNA-dependent protein kinase catalytic subunit,DNA-PKcs
ORGANELLEORCELLULARCOMPONENT
#1
1
NATURAL
3
X-ray repair cross-complementing protein 5
ORGANELLEORCELLULARCOMPONENT
#2
1
RECOMBINANT
Molecular weight
Value: 0.48 MDa / Experimental value: NO
Source (natural)
ID
Entity assembly-ID
Organism
Ncbi tax-ID
1
2
Homo sapiens (human)
9606
2
3
Homo sapiens (human)
9606
Source (recombinant)
Organism: Escherichia coli (E. coli)
Buffer solution
pH: 7.4
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Vitrification
Cryogen name: ETHANE
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELDBright-field microscopy
Image recording
Electron dose: 54.49 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
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Processing
EM software
ID
Name
Category
1
Warp
particleselection
2
EPU
imageacquisition
13
cryoSPARC
3Dreconstruction
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 104374
Symmetry
Point symmetry: C1 (asymmetric)
3D reconstruction
Resolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43995 / Symmetry type: POINT
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