Ku70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of the Ku ring along DNA.

Non-homologous end-joining (NHEJ) is a major double-stranded DNA (dsDNA) break repair pathway essential for V(D)J recombination during lymphocyte development. The Ku70/Ku80 heterodimer (Ku) initiates NHEJ by encircling dsDNA ends and recruiting DNA-PKcs. Ku70 in plants and mammals acquired a C-terminal SAP domain implicated in nucleic acid binding. Here, we show that in murine models, the SAP domain is dispensable for Ku stability and recruitment to DNA breaks. Unlike Ku70-/- mice, Ku70ΔSAP/ΔSAP mice exhibit normal lymphocyte development despite mild radiation sensitivity. Structural modeling places the SAP domain in adjacent DNA grooves, where it can restrict Ku's lateral movement along dsDNA. Correspondingly, in mice lacking DNA-PKcs that caps the ends, Ku70ΔSAP reduces T cell counts and deletion sizes, consistent with Ku translocating off DNA. Moreover, SAP deletion reduced DNA-end affinity, increased dissociation, and exchange of purified Ku at low concentrations, and increased multiple-loading at high concentrations, consistent with increased lateral movement. In DNA-PKcs-/- murine fibroblasts, deletion or lysine mutation (K593/4A, corresponding to K595/6A in human Ku70) in the SAP domain decreased the relative intensity of laser-induced Ku spots, revealing a role of the SAP domain in constraining Ku lateral movement on dsDNA in the absence of DNA-PKcs (or in the short-range complex).