Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enor mous, 470-kDa protein serine/threonine kinase that has homology with member s of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contr ibutes to the repair of DNA double-strand breaks (DSBs) by assembling broke n ends of DNA molecules in combination with the DNA-binding factors Ku70 an d Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the rol e of protein kinase activity in the repair of DNA DSBs. We constructed a co ntiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient i n DNA-PKcs (M059J and V3). We then created deletion and site-directed mutat ions within the conserved PI 3-kinase domain of the DNA-PKcs gene to test t he importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement th e DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These re sults indicate that the protein kinase activity of DNA-PKcs is essential fo r the rejoining of DNA DSBs in mammalian cells. We have also determined a m odel structure for the DNA-PKcs kinase domain based on comparisons to the c rystallographic structure of a cyclic AMP-dependent protein kinase. This st ructure gives some insight into which amino acid residues are crucial for t he kinase activity in DNA-PKcs.