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 DNA-dependent protein kinase catalytic subunits (DNA-PKcs) that caps the ends. To investigate the function of Ku70 C-terminal SAP domain, we generated a mouse model with knock-in deletion (Ku70delSAP/delSAP). Ku70delSAP supports KU stability and its recruitment to DNA damage sites. Contrary to the growth retardation and immunodeficiency of Ku70-/- mice, Ku70delSAP/delSAP mice have normal size and lymphocyte development. Structural modeling of KU on long dsDNA suggests that the SAP domain binds to an adjacent major groove and potentially limits KU rotation and lateral movement on dsDNA. Accordingly, with the loss of DNA-PKcs that caps the ends, Ku70delSAP fails to form stable damage foci. In DNA-PKcs-/-mice, Ku70delSAP abrogates the leaky T-cell development and compromises residual end-joining in vivo. In the absence of DNA-PKcs, purified Ku70delSAP has reduced affinity for DNA ends, dissociates more readily at lower concentrations, and accumulates as multimers at high concentrations. These findings revealed the role of the KU SAP domain in restricting KU rotation and lateral movement on DNA that is largely masked by DNA-PKcs.