DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair

Vertebrate cells have evolved two major pathways for repairing DNA double-s trand breaks (DSBs), homologous recombination (HR) and nonhomologous DNA en d-joining (NHEJ). To investigate the role of DNA ligase IV (Lig4) in DSB re pair, we knocked out the Lig4 gene (LIG4) in the DT40 chicken B-lymphocyte cell line. The LIG4(-/-) cells showed a marked sensitivity to X-rays, bleom ycin, and VP-16 and were more x-ray-sensitive in G, than late S or G(2)/M, suggesting a critical role of Lig4 in DSB repair by NHEJ In support of this notion, HR was not impaired in LIG4(-/-) cells. LIG4(-/-) cells were more x-ray-sensitive when compared with KU70(-/-) DT40 cells, particularly at hi gh doses. Strikingly, however, the x-ray sensitivity of KU70(-/-)/LIG4(-/-) double-mutant cells was essentially the same as that of KU70(-/-) cells, s howing that Lig4 deficiency has no effect in the absence of Ku. These resul ts indicate that Lig4 is exclusively required for the Ku-dependent NHEJ pat hway of DSB repair and that other DNA ligases (I and III) do not substitute for this function. Our data may explain the observed severe phenotype of L ig4-deficient mice as compared with Ku-deficient mice.