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 G2͞M, 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.

Hypersensitivity of Nonhomologous DNA End-joining Mutants to VP-16 and ICRF-193

“…RAD54 Ϫ/Ϫ cells are defective in HR and also display increased radiosensitivity, and RAD54 Ϫ/Ϫ /KU70 Ϫ/Ϫ cells, defective in both HR and NHEJ, are much more radiosensitive than each single mutant (18). Interestingly, the extent of radiosensitivity of RAD54 Ϫ/Ϫ cells is very similar to that of LIG4 Ϫ/Ϫ cells (and also DNA-PKcs Ϫ/Ϫ/Ϫ cells) (17,18,23), indicating that the two repair pathways, HR and NHEJ, contribute equally to the repair of DSBs in the DT40 cell line.We previously showed that LIG4 Ϫ/Ϫ cells were more VP-16-sensitive than wild-type cells (17). This result was simply interpreted as a DSB repair defect of the LIG4 Ϫ/Ϫ cells, based on the fact that VP-16 is a topo II poison that generates DSBs.…”

“…Finally, the DNA ligase IV-Xrcc4 complex is recruited for ligation. The requirement for DNA ligase IV in this pathway is exclusive, as other DNA ligases (I and III) are unable to substitute for the ligase IV function (16,17).Consistent with the proposed functions of HR and NHEJ in DSB repair, cells deficient in HR or NHEJ proteins have been shown to be highly sensitive to DSB-generating DNA-damaging agents, such as ionizing radiation (16 -23). In a chicken B-lymphocyte DT40 cell line, where HR activity is extraordinarily high as compared with other vertebrate cell lines (24), several knockout mutants deficient in HR and/or NHEJ have been constructed by gene targeting.…”

“…Indeed, the S. cerevisiae rad52 mutant is extremely sensitive to topo II poisons (43). In higher eukaryotes, several NHEJ mutants are shown to exhibit hypersensitivity to topo II poisons (17,44,45).Much less is known about the mechanisms for the repair of ICRF-193-induced topo II clamps, assuming that these intermediates are actually cytotoxic DNA damage. Importantly, however, recent work has revealed that ICRF-193-induced topo II clamps also undergo 26 S proteasome-mediated degradation (46), raising an intriguing possibility that the repair machinery responsible for cleavable complexes are, at least in part, involved in the repair of topo II clamps as well.…”

“…Finally, the DNA ligase IV-Xrcc4 complex is recruited for ligation. The requirement for DNA ligase IV in this pathway is exclusive, as other DNA ligases (I and III) are unable to substitute for the ligase IV function (16,17).…”

“…Indeed, the S. cerevisiae rad52 mutant is extremely sensitive to topo II poisons (43). In higher eukaryotes, several NHEJ mutants are shown to exhibit hypersensitivity to topo II poisons (17,44,45).…”