DNA-DEPENDENT PROTEIN-KINASE - DNA-BINDING AND ACTIVATION IN THE ABSENCE OF KU

In mammalian cells, double-strand break repair and V(D)J recombination require DNA-dependent protein kinase (DNA-PK), a serine/threonine kin ase that is activated by DNA. DNA-PK consists of a 460-kDa subunit (p4 60) that contains a putative kinase domain and a heterodimeric subunit (Ku) that binds to double-stranded DNA ends. Previous reports suggest ed that the activation of DNA-PK requires the binding of Ku to DNA, To investigate this further, p460 and Ku were purified separately to hom ogeneity, Surprisingly, p460 was capable of binding to DNA in the abse nce of Ku, The binding of p460 to double-stranded DNA ends was salt-la bile and could be disrupted by single-stranded or supercoiled DNA, pro perties distinct from the binding of Ku to DNA, Under low salt conditi ons, which permitted the binding of p460 to DNA ends, the kinase was a ctivated. Under higher salt conditions, which inhibited the binding of p460, activation of the kinase required the addition of Ku, Significa ntly, when the length of DNA decreased to 22 bp, Ku competed with p460 for DNA binding and inhibited kinase activity, These data demonstrate that p460 is a self-contained kinase that is activated by direct inte raction with double-stranded DNA and that the role of Ku is to stabili ze the binding of p460 to DNA ends.