Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.