The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought t o target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA -PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subu nit of Ku, and shown that the C-terminal 178 amino acid residues are dispen sable for DNA end-binding activity but are required for efficient interacti on of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the termin al 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J re combination. These cells have therefore acquired the phenotype of mouse SCI D cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is requi red for DNA double-strand break rejoining and coding but not signal joint f ormation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This r esidue is conserved among species and lies outside of the previously report ed Ku70-Ku80 interaction domain. The mutational change nonetheless abrogate s the Ku70-Ku80 interaction and DNA end-binding activity.