National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM135651
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
U24GM129547
United States
Citation
Journal: Sci Adv / Year: 2023 Title: Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ. Authors: Siyu Chen / Alex Vogt / Linda Lee / Tasmin Naila / Ryan McKeown / Alan E Tomkinson / Susan P Lees-Miller / Yuan He / Abstract: DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). ...DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)-free, short-range state to align the DSB ends (Chen ). Using single-particle cryo-electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.
UniProtKB: DNA-dependent protein kinase catalytic subunit
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
1.3 mg/mL
Buffer
pH: 7.4
Grid
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 302 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
TFS KRIOS
Temperature
Min: 70.0 K / Max: 70.0 K
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 13132 / Average exposure time: 4.0 sec. / Average electron dose: 65.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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