Critical residues in the Ku70 von Willebrand A domain mediate Ku interaction with the LigIV-XRCC4 complex in non-homologous end-joining

The Ku heterodimer (Ku70/Ku80) is central to the non-homologous end-joining (NHEJ) pathway. Ku binds to the broken DNA ends and promotes the assembly of the DNA repair complex. The N-terminal Ku70 von Willebrand A (vWA) domain is known to mediate protein-protein interactions important for the repair process. In particular, the D192 and D195 residues within helix 5 of the Ku70 vWA domain were shown to be essential for NHEJ function, although the precise role of these residues was not identified. Here, we set up a miniTurbo screening system to identify Ku70 D192/D195 residue-specific interactors in a conditional, human Ku70-knockout cell line in response to DNA damage. Using fusion protein constructs of Ku70 wild-type and mutant (D192A/D195R) with miniTurbo, we identified a number of candidate proximal interactors in response to DNA damage treatment, including DNA Ligase IV (LigIV), a known and essential NHEJ complex member. Interestingly, LigIV was enriched in our wildtype screen but not the Ku70 D192A/D195R screen, suggesting its interaction is disrupted by the mutation. Validation experiments demonstrated that the DNA damage-induced interaction between Ku70 and LigIV was disrupted by the Ku70 D192A/D195R mutations. Our findings provide greater detail about the interaction surface between the Ku70 vWA domain and LigIV and offer strong evidence that the D192 and D195 residues are important for NHEJ completion through an interaction with LigIV. Altogether, this work reveals novel potential proximal interactors of Ku in response to DNA damage and identifies Ku70 D192/D195 residues as essential for LigIV interaction with Ku during NHEJ.