Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ

Chen, Siyu; Vogt, Alex; Lee, Linda; Naila, Tasmin; McKeown, Ryan; Tomkinson, Alan E.; Lees-Miller, Susan P.; He, Yuan

doi:10.1126/sciadv.adg2838

Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ

Journal Article · Wed May 31 00:00:00 EDT 2023 · Science Advances

DOI:https://doi.org/10.1126/sciadv.adg2838· OSTI ID:2424151

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Northwestern Univ., Evanston, IL (United States)
Univ. of Calgary, AB (Canada); Alberta Precision Laboratories, Calgary, AB (Canada)
Univ. of New Mexico, Albuquerque, NM (United States)
Univ. of Calgary, AB (Canada)

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 et al.). 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 et al.). 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.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 2424151

Journal Information:: Science Advances, Journal Name: Science Advances Journal Issue: 22 Vol. 9; ISSN 2375-2548

Publisher:: AAASCopyright Statement

Country of Publication:: United States

Language:: English

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