Ku and DNA‐PK dynamic conformations and assembly regulate DNA binding and the initial NHEJ complex

DNA double‐strand break (DSB) repair by non‐homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA‐PKcs solution structures alone and in complex with DNA, defined by X‐ray scattering (SAXS), reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C‐terminal region forms a flexible arm that extends from the DNA‐binding core to recruit and retain DNA‐PKcs at DSBs. Furthermore, Ku and DNA promoted assembly of a DNA‐PKcs dimer facilitates trans‐autophosphorylation at the DSB. The resulting site‐specific autophosphorylation induces a large conformational change that opens DNA‐PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA‐PKcs rearrangements to control DNA‐PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.