Late embryonic lethality and impaired V (D)J recombination in mice lacking DNA ligase IV

Abstract

The DNA-end-joining reactions used for repair of double-strand breaks in DNA and for V(D)J recombination, the process by which immunoglobulin and T-cell antigen-receptor genes are assembled from multiple gene segments, use common factors. These factors include components of DNA-dependent protein kinase (DNA-PK), namely DNA-PKcs and the Ku heterodimer, Ku70-Ku80, and XRCC4. The precise function of XRCC4 is unknown, but it interacts with DNA ligase IV. Ligase IV is one of the three known mammalian DNA ligases; however, the in vivo functions of these ligases have not been determined unequivocally. Here we show that inactivation of the ligase IV gene in mice leads to late embryonic lethality. Lymphopoiesis in these mice is blocked and V(D)J joining does not occur. Ligase IV-deficient embryonic fibroblasts also show marked sensitivity to ionizing radiation, growth defects and premature senescence. All of these phenotypic characteristics, except embryonic lethality, resemble those associated with Ku70 and Ku80 deficiencies, indicating that they may result from an impaired end-joining process that involves both Ku subunits and ligase IV. However, Ku-deficient mice are viable, so ligase IV must also be required for processes and/or in cell types in which Ku is dispensable.

DNA double-strand break repair and development

“…Xrcc4 À/À and Lig4 À/À mice are virtually identical in phenotype, and in contrast to the viability of DNA-PK-deficient mice, are embryonic lethal, presumably due to abundant apoptosis of newly postmitotic neurons (Barnes et al, 1998;Frank et al, 1998;Gao et al, 1998b). Like other NHEJ-deficient models, Xrcc4 À/À and Lig4 À/À embryos also showed impaired V(D)J recombination and embryonic fibroblasts exhibited IR sensitivity and premature senescence (Frank et al, 1998;Gao et al, 1998b). Ku70 À/À and Ku80 À/À mice, while viable, likewise showed increased apoptosis in post-mitotic neural cells, though to a lesser extent than seen in Xrcc4 À/À and Lig4 À/À embryos (Gu et al, 2000).…”

“…The extent to which Artemis is required during canonical NHEJ (independent of V(D)J recombination) remains uncertain, as conflicting data on its requirement for end joining have been reported (Rooney et al, 2003;Riballo et al, 2004;Zhang et al, 2004;Wang et al, 2005b). However, Lig4 and Xrcc4 are essential for NHEJ and embryonic survival (Barnes et al, 1998;Frank et al, 1998;Gao et al, 1998b), and their interaction is required for efficient end joining as Lig4 is not sufficient to complete repair in the absence of Xrcc4 (Critchlow et al, 1997;Grawunder et al, 1997Grawunder et al, , 1998aBassing and Alt, 2004). While HR is available for repair during late S/G 2 , NHEJ is most prominently activated in G 1 , G 0 and early S phase of the cell cycle (Rothkamm et al, 2003;Thacker and Zdzienicka, 2004).…”

Reprint of “The clinical impact of deficiency in DNA non-homologous end-joining”

“…Disruption of the DNA ligase IV (Lig4) gene (resulting in complete loss of DNA ligase IV function) causes embryonic lethality in mice with extensive neuronal apoptosis around E13.5 [40][41][42]. Thus, DNA ligase IV is essential for embryonic viability.…”

Non-homologous end-joining, a sticky affair

“…NHEJ efficiency is minimal in the absence of this complex and a defect in ligase IV or XRCC4 is even lethal in mice (Barnes et al, 1998;Frank et al, 1998;Gao et al, 1998b). XRCC4 is absolutely required for ligase IV activity, probably because the ligase IV protein needs it for stability and correct targeting to DSBs (Grawunder et al, 1997;Bryans et al, 1999;Chen et al, 2000;Teo and Jackson, 2000;Hsu et al, 2002;Drouet et al, 2005;Costantini et al, 2007).…”

“…NHEJ is critical for differentiating cells to prevent apoptosis of the post-mitotic neurons (Lee and McKinnon, 2007). This is particularly clear in DNA ligase IV and XRCC4-deficient mice (Frank et al, 1998;Gao et al, 1998b), but Ku70 or Ku80 deficiency shows similar defects to a lesser extent (Nussenzweig et al, 1996;Gu et al, 1997). The neuropathy in patients with the LIG4 syndrome and XLF/Cernunnos deficiency is confined to characteristic facial features and microcephaly, suggesting that these mutations do not cause a complete NHEJ deficiency.…”