The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication

Exposure of cells to ionizing radiation inhibits DNA replication in a dose- dependent manner, The dose response is biphasic and the initial steep compo nent reflects inhibition of replicon initiation thought to be mediated by a ctivation of the S-phase checkpoint. In mammalian cells, inhibition of repl icon initiation requires the ataxia telagiectasia mutated (ATM) gene, a mem ber of the phosphatidyl inositol kinase-like (PIKL) family of protein kinas es, We studied the effect on replicon initiation of another member of the P I-3 family of protein kinases, the catalytic subunit of DNA-dependent prote in kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distr ibution of nascent DNA using alkaline sucrose gradient centrifugation. Expo sure of human cells proficient in DNA-PKcs (HeLa or M059-K) to 10 Gy inhibi ted replicon initiation in a time-dependent manner, Inhibition was at a max imum 1 h after irradiation and recovered at later times, Similar treatment of human cells deficient in DNA-PKcs (M059-J) inhibited replicon initiation to a similar level and with similar kinetics; however, no evidence for rec overy, or only limited recovery, was observed for up to 8 h after irradiati on. In addition a defect was observed in the maturation of nascent DNA, Sim ilarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed l ittle evidence for recovery of DNA replication inhibition up to 6 h after i rradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h, Normal kinetics of recovery were observed in xrs-5 cells, deficient i n Ku80; in 180BR cells, deficient in DNA ligase IV; as well as XR-1 cells, deficient in XRCC4, an accessory factor of DNA ligase IV. Since all these c ell lines share the DNA double strand break rejoining defect of M059-J and irs20 cells, the lack of recovery of DNA replication in the latter cells ma y not be attributed entirely to the prolonged presence of unrepaired DNA ds b, We propose that DNA-PKcs, in addition to its functions in the rejoining of DNA dsb and in DNA replication, also operates in a pathway that in norma l cells facilitates recovery of DNA replication after irradiation.