The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations - PubMed (original) (raw)

The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations

D O Ferguson et al. Proc Natl Acad Sci U S A. 2000.

Abstract

We have used spectral karyotyping to assess potential roles of three different components of the nonhomologous DNA end-joining pathway in the maintenance of genomic stability in mouse embryonic fibroblasts (MEFs). MEFs homozygous for mutations that inactivate either DNA ligase IV (Lig4) or Ku70 display dramatic genomic instability, even in the absence of exogenous DNA damaging agents. These aberrant events range from chromosomal fragmentation to nonreciprocal translocations that can involve several chromosomes. DNA-dependent protein kinase catalytic subunit deficiency also promotes genome instability. Deficiency for the p53 cell cycle checkpoint protein has little effect on spontaneous levels of chromosomal instability in Lig4-deficient fibroblasts. However, in the context of ionizing radiation treatment, p53 deficiency allowed visualization of massive acute chromosomal destruction in Lig4-deficient MEFs, which in surviving cells manifested as frequent nonreciprocal translocations. We conclude that nonhomologous DNA end-joining plays a crucial role as a caretaker of the mammalian genome, and that an alternative repair pathway exists that often leads to nonreciprocal translocations.

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Figures

Figure 1

Figure 1

Spontaneous chromosomal fragmentation in NHEJ-deficient MEFs. Arrows indicate the aberration. (a_–_c) Images of 4′,6-diamidino-2-phenylindole (DAPI)-stained metaphase chromosomes. (d) Both DAPI staining and SKY analysis. (a) _Lig4_−/−; broken chromatid. (b) _DNA-PKcs_−/−; two marker chromosomes (acentric fragments). (c) _Ku70_−/−; single chromatid gap. (d) _Ku70_−/−; double chromatid break. (Left) DAPI stain; (Right) SKY analysis. The fragment and the adjacent chromosome show identical staining by SKY, suggesting that the fragment arose from the termini of the adjacent chromosome 7.

Figure 2

Figure 2

Spontaneous nonreciprocal translocations arising in NHEJ-deficient cells. (a) _Lig4_−/− MEF. An unusual chromatid interaction is seen by DAPI staining (Left), which is comprised of at least two chromosomes visible after hybridization with the SKY probe mixture (Center). Spectral analysis yields classified colors (Right), indicating that this is a complex translocation involving material from three different chromosomes. From left to right, the chromosomal material is 15, 8, and X. (b) The cell of origin was from a dispersed abdominal organ block of a midgestation _Lig4_−/− embryo. The cell was not cultured or exposed to colcemid. The exact cell type is not known. DAPI staining (Left), visible colors after SKY probe hybridization (Center), and spectral analysis (Right) are shown. The chromosome arose from a simple t(5:6) translocation. (c) _Lig4_−/−_p53_−/− MEF. Complex translocation involving material from as many as four different chromosomes. DAPI staining (Left), visible colors after SKY hybridization (Center), and classified colors after spectral analysis (Right) are shown. The chromosomal material, from top to bottom, is 13, 7, 17, and 11.

Figure 3

Figure 3

Genomic instability induced by IR. (a) Portion of a metaphase spread from a wild-type MEF 24 h after a 500-rad dose of IR. The arrow indicates an example of a fragment. (b) Portion of a metaphase from a Lig4p53 double mutant MEF 24 h after a 500-rad dose of IR. Innumerable chromosomal aberrations are evident. (c) Reciprocal translocation involving chromosome 2 (red) and chromosome 7 (pink) found in a wild-type metaphase 12 days after irradiation. DAPI (Left), spectral (Center), and classified (Right) displays are shown for both translocated chromosomes. (d) Portion of a metaphase (spectral colors) from an irradiated _Lig4_−/−_p53_−/− culture 12 days after irradiation. Two nonreciprocal translocations are denoted by the arrowheads.

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