biostudies-literatureChen SNCI NIH HHSNIGMS NIH HHSeadg2838https://www.ebi.ac.uk/biostudies/studies/S-EPMC10413680biostudies-literatureUnknown9(22)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 <i>et al.</i>). 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 <i>et al.</i>). 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 DNScience advancesCryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.PMC10413680R01 GM144559T32 GM008382U24 GM129547P30 CA060553R01 GM047251P01 CA092584P30 CA118100R01 GM135651T32 GM140995McKeown RHe YLee LTomkinson AENaila TVogt ALees-Miller SPChen SfalseCryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.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 <i>et al.</i>). 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 <i>et al.</i>). 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 DN2023-01-01T00:00:00Z2023 Jun2026-05-29T07:39:02.307Z2025-02-19T01:18:12.331ZS-EPMC104136803725694710.1126/sciadv.adg2838