MOLECULAR AND BIOCHEMICAL-CHARACTERIZATION OF DNA-DEPENDENT PROTEIN KINASE-DEFECTIVE RODENT MUTANT IRS-20

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) i s a member of a sub-family of phosphatidylinositol (PI) 3-kinases term ed PIK-related kinases, A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-ki nase domain. Mutants defective in DNA-PKcs are sensitive to ionising r adiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than tw o previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but i s unable to function as a protein kinase. To verify the defect in irs- 20 cells and provide insight into the function and expression of DNA-P Kcs in double-strand break repair and V(D)J recombination we introduce d YACs encoding human and mouse DNA-PKcs into defective mutants and ac hieved complementation of the defective phenotypes. Furthermore, in ir s-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus . This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is imp ortant for protein kinase activity.