The dna double-strand break repair mutant cell line irs-20 expresses an inactive dna-dependent protein kinase catalytic subunit

The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460kD catalytic subunit (DNA-PKcs) and a heterodimeric DNA-binding complex comprised of the 70 and 86 kD Ku autoantigen proteins. Mutations that affect the expression of the Ku 80 or catalytic subunit of DNA-PK (DNA-PKcs) disrupt V(D)J recombination and DNA double-strand break repair pathways. Here, we show the radiosensitive rodent cell line IRS-20 contains a defect that disrupts the expression and kinase activity of DNA-PK. Expression of the DNA-PKcs is reduced four-fold in the IRS-20 cells relative to the parental cell 10B2 and the IRS-20 enzyme is inactive using recombinant RPA as an in vitro substrate for kinase activity. Human chromosome 8, which contains the gene encoding the human DNA-PKcs, complements the radiosensitivity defect of the IRS-20 cells and restores DNA-PK kinase activity. These results suggest the kinase activity of DNA-PK is neccessary for DNA double-stranded break repair and support a model whereby DNA-PK regulates double-stranded DNA break repair via the phosphorylation of protein substrates, some of which may directly participate in this DNA-repair pathway.