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-strand breaks (DSBs), homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ). To investigate the role of DNA ligase IV (Lig4) in DSB repair, 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, bleomycin, and VP-16 and were more x-ray-sensitive in G(1) 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 high doses. Strikingly, however, the x-ray sensitivity of KU70(-/-)/LIG4(-/-) double-mutant cells was essentially the same as that of KU70(-/-) cells, showing that Lig4 deficiency has no effect in the absence of Ku. These results indicate that Lig4 is exclusively required for the Ku-dependent NHEJ pathway 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 Lig4-deficient mice as compared with Ku-deficient mice.
Adachi
N
N
Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Totsuka-ku, Yokohama 244-0813, USA.
Ishino
T
T
Ishii
Y
Y
Takeda
S
S
Koyama
H
H
D000818:Animals; D034961:Antigens, Nuclear; D001402:B-Lymphocytes; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D004249:DNA Damage; D004265:DNA Helicases; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011839:Radiation, Ionizing
D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP