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

Abstract

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.

Figure 1

Generation of LIG4^−/− clones. (A) Schematic representation of targeted disruption of the chicken LIG4 gene. The chicken LIG4 locus, two targeting constructs (pLIV-hyg and pLIV-pur), and the targeted locus are shown. The black box indicates the ORF of the LIG4 gene. The triangles flanking the hygromycin resistance (hyg^r) or puromycin resistance (pur^r) gene designate loxP sequences. (B) Southern blot analysis. EcoRI-digested genomic DNA of wild-type (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) cells was hybridized with the probe shown in A as described. (C) RT-PCR analysis. Total RNA of wild-type, LIG4^+/−, and LIG4^−/− cells was used as a template to amplify a LIG4 cDNA.

Figure 2

Increased sensitivity of LIG4^−/− cells to DSBs. (A) Growth curves of wild-type and LIG4^−/− cells. Data are representative of three independent experiments. (B) Sensitivity of cells to ionizing radiation. (C) Sensitivity of cells to bleomycin. (D) Sensitivity of cells to VP-16. (E) X-ray sensitivity of cells throughout the cell cycle. Wild-type, LIG4^−/−, and KU70^−/− cells were synchronized in G₁ with 0.4 mM mimosine. After release from mimosine treatment, the cells were x-irradiated (2 Gy) at the indicated time points and were subjected to a colony formation assay. Data are representative of two independent experiments. (A–D) Data are the mean of three independent experiments.

Figure 3

LIG4^−/− cells are more x-ray-sensitive than KU70^−/− and KU70^−/−/LIG4^−/− cells. (A) X-ray sensitivity of wild-type, LIG4^−/−, KU70^−/−, and KU70^−/−/LIG4^−/− cells. Data are representative of two independent experiments. (B) X-ray sensitivity of KU70^−/−/LIG4^−/− cell lines stably transfected with an expression plasmid for chicken Ku70 (6).

Figure 4

Model for DSB repair pathways. DSBs can be repaired by either NHEJ (A) or HR (B). However, once Ku binds to DSB ends, the DSBs must be repaired by NHEJ, preventing the opportunity to use HR. Because Lig4 is exclusively required for the Ku-dependent NHEJ pathway, its absence results in unrepaired lesions, leading to cell death (C). At least in DT40 cells, Ku deficiency is not so severe, presumably because HR is able to partially compensate for NHEJ.