Role of DNA-dependent protein kinase in recognition of radiation-induced DNA damage in human peripheral blood mononuclear cells

The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in r adiation-induced DNA damage recognition. The complex includes the ku hetero dimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKcs, Th e activation of the DNA-PK complex was studied in X-irradiated peripheral b lood mononuclear cells (PBMC) from subjects of different ages. Radiation-in duced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were det ermined in nuclear and cytoplasmic extracts. DNA-binding activity was incre ased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extra cts regardless of age. The radiation-induced activation of the DNA-PK compl ex may result from the increased concentrations of ku 80 and DNA-PKcs in th e cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These f indings suggest major steps involved in DNA-PK activation, and the intracel lular and molecular changes that may account for the age-dependent impairme nt of DNA repair capacity in irradiated mammalian cells.