Differential Phosphorylation of DNA-PKcs Regulates the Interplay between End-Processing and End-Ligation during Nonhomologous End-Joining

Nonhomologous end-joining (NHEJ) is a major DNA double-strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate-specific NHEJ factor that can be autophosphorylated or transphosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated transphosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strict autophosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcsKD/KD mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations. [Display omitted] •DNA-PKcs protein inhibits direct end-ligation at the DNA ends•Intermolecular autophosphorylation of DNA-PKcs is required for end-ligation•DNA-PKcs protein, but not its kinase activity, is required for end-processing•ATM phosphorylates DNA-PKcs to recruit Artemis and promote end-processing Differential phosphorylation of DNA-PKcs at DNA ends is the molecular switch between end-processing and end-ligation. ATM-mediated phosphorylation of DNA-PKcs is necessary for Artemis recruitment and end-processing, while intermolecular autophosphorylation of DNA-PKcs relieves the physical block on end-ligation imposed by the DNA-PKcs protein at DNA ends and is required for direct end-ligation.