Genetic analysis of the DNA-dependent protein kinase reveals an inhibitoryrole of Ku in late S-G(2) phase DNA double-strand break repair

Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-h omologous end joining, which requires the DNA-dependent protein kinase (DNA -PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku7 0 and Ku80 heterodimer. To define the activities of individual DNA-PK compo nents in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. D NA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppr essed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when s ister chromatids are available for HR repair. To test whether direct interf erence by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radiores istance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restricti on enzyme was increased in the absence of Ku but not of DNA-PKcs. Significa ntly, abrogation of HR activity by targeting RAD54 in Ku 70(-/-) or DNA-PKc s(-/-/-) cells caused extreme radiosensitivity, suggesting that the relativ e radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.