Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.