Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination

DNA-dependent protein kinase (DNA-PK) is utilized in both BRA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which th is multiprotein complex participates in these processes is unknown, To eval uate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3 -kinase inhibitor wortmannin on the repair of ionizing radiation-induced DN A double-strand breaks and V(D)J recombination in the V(D)J recombinase ind ucible B cell line HDR37, Wortmannin radiosensitized HDR37, but had no affe ct on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wildtype hum an DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstra ted that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombinatio n. These apparently contradictory results were reconciled by analyses of ce ll lines varying in their expression of recombinant wild-type human DNA-PKc s, These studies establish that minimal DNA-PKcs protein levels are suffici ent to support V(D)J recombination, but insufficient to confer resistance t o ionizing radiation.