Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining.

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.