The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA
The non-homologous end-joining (NHEJ) pathway is critical for DNA double-strand break repair and is essential for lymphocyte development and maturation. The Ku70/Ku80 heterodimer (KU) binds to DNA ends, initiating NHEJ and recruiting additional factors, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that caps the ends and pushes KU inward. The C-terminus of Ku70 in higher eukaryotes includes a flexible linker and a SAP domain, whose physiological role remains poorly understood. To investigate this, we generated a mouse model with knock-in deletion of the SAP domain (Ku70DSAP/DSAP). Ku70DSAP supports KU stability and its recruitment to DNA damage sites in vivo. In contrast to the growth retardation and immunodeficiency seen in Ku70-/- mice, Ku70DSAP/DSAP mice show no defects in lymphocyte development and maturation. Structural modeling of KU on long dsDNA, but not dsRNA suggests that the SAP domain can bind to an adjacent major groove, where it can limit KU's rotation and lateral movement along the dsDNA. Consistent with this model, in the absence of DNA-PKcs that caps the ends, Ku70DSAP fails to support stable DNA damage-induced KU foci. In DNA-PKcs-/- mice, Ku70DSAP abrogates the leaky T cell development and reduces both the qualitative and quantitative aspects of residual V(D)J recombination. Moreover, purified Ku70DSAP also has reduced affinity for DNA ends and dissociates more readily. These findings revealed a physiological role of the SAP domain in NHEJ by restricting KU rotation and lateral movement on DNA that is largely masked by DNA-PKcs. Overall design: To elucidate the function of the KU70 SAP domain in vivo, we generated a mouse model with targeted deletion of the aa 558-608, encompassing the entire SAP domain of endogenous Ku70 while sparing the NLS. According to High Throughput Genomic Translocation (HTGT)-Seq, both V(D)J recombination and CSR, which entirely depend on KU and NHEJ, remain normal in Ku70DSAP/DSAP cells. Ku70DSAP reduced the residual SJs and leaking CJs in DNA-PKcs-/- lymphocytes and decreased the deletion size in V(D)J junctions recovered from DNA-PKcs-/- cells.