Updated project metadata.
DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damages as they can lead to mutations and chromosomal rearrangements, which underlie cancer development and progression. Non-homologous end-joining (NHEJ) is the dominant pathway for the repair of DSBs in human cells, which involves the DNA binding proteins Ku70/Ku80. Other DNA binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair. However, their role in NHEJ has remained elusive. Here we show that ZNF384, which is a member of the C2H2 family of ZnF proteins that binds DNA in vitro, is recruited to DSBs in vivo. ZNF384 recruitment requires the PARP/PAR-dependent expansion of damaged chromatin followed by binding of ZNF384 to the exposed DNA via its unique C2H2 motifs. Mass-spectrometry-based experiments revealed that ZNF384 interacts with the Ku70/Ku80 NHEJ complex via its N-terminus. This interaction promotes the assembly of Ku70/Ku80 at DBSs as revealed by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. By regulating Ku70/80 dynamics, ZNF384 facilitates the assembly of several downstream NHEJ factors (e.g. APLF and XRCC4) and repair by cNHEJ at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds to DSBs and Ku70/80 to facilitate their binding and the subsequent build-up of a functional cNHEJ repairosome at these lesions, high-lighting a role for ZNF384 in DSB repair and genome maintenance.