Postreplicative Joining of DNA Double-Strand Breaks Causes Genomic Instability in DNA-PKcs–Deficient Mouse Embryonic Fibroblasts

Abstract

Combined cytogenetic and biochemical approaches were used to investigate the contributions of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the maintenance of genomic stability in nonirradiated and irradiated primary mouse embryo fibroblasts (MEF). We show that telomere dysfunction contributes only marginally to genomic instability associated with DNA-PKcs deficiency in the absence of radiation. Following exposure to ionizing radiation, DNAPKcs À/À MEFs are radiosensitized mainly as a result of the associated DNA double-strand break (DSB) repair defect. This defect manifests as an increase in the fraction of DSB rejoining with slow kinetics although nearly complete rejoining is achieved within 48 hours. Fifty-four hours after ionizing radiation, DNA-PKcs À/À cells present with a high number of simple and complex chromosome rearrangements as well as with unrepaired chromosome breaks. Overall, induction of chromosome aberrations is 6-fold higher in DNA-PKcs À/À MEFs than in their wild-type counterparts. Spectral karyotypingfluorescence in situ hybridization technology distinguishes between rearrangements formed by prereplicative and postreplicative DSB rejoining and identifies sister chromatid fusion as a significant source of genomic instability and radiation sensitivity in DNA-PKcs À/À MEFs. Because DNA-PKcsMEFs show a strong G 1 checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs À/À MEFs prolongs the mean life of broken chromosome ends and increases the probability of incorrect joining. The preponderance of sister chromatid fusion as a product of incorrect joining points to a possible defect in S-phase arrest and emphasizes proximity in these misrepair events. (Cancer Res 2005; 65(22): 10223-32)

Telomere dysfunction: a new player in radiation sensitivity

“…(54) A similar although less pronounced situation was observed in primary cells of mice with a null mutation for DNA-PKcs. (55) Telomere-DSB fusions with interstitial telomeric signals at the junction point were observed in primary embryonic fibroblasts derived from this DNA-PKcs-deficient mice model, reinforcing the idea that not only eroded but also structurally dysfunctional telomeres can join radiation or spontaneous broken chromosome ends (Fig. 3).…”

Distribution of Telomeric DNA Sequences on the X-Radiation-Induced Chromosome Fragments Observed in the Genome of Androgenetic Brook Trout (<b><i>Salvelinus fontinalis</i></b>, Mitchill 1814)

“…Chromosome fragments, showing 2 always terminally located telomeres, represented another chromatin arrangement. Such shape and distribution of the hybridization signals suggested a formation in the course of telomere loss in just one chromosome arm and fusion between unprotected sister chromatids only [Bolzan and Bianchi, 2006;Martín et al, 2005]. On the other hand, broken chromosomal ends might have been repaired via 2 mechanisms: (i) addition of telomere DNA sequences to the broken chromatid ends of one arm, while (ii) broken chromatid ends of the other arm may undergo fusion.…”

“…In the chromosome fragments with fused sister chromatids, breakages accompanying separation of the chromatids during anaphase when 2 kinetochores are pulled in the opposite directions may result in formation of the subsequent acentric and centric fragments [Martín et al, 2005]. This could partially explain intraindividual variation in the chromosome fragment number and size observed in the androgenetic fish.…”

ATM and DNA-PKcs make a complementary couple in DNA double strand break repair

“…Nonetheless, animal [32] and human cell lines which completely lack DNA-PKcs protein have been artificially generated, and they all present proliferation and genome stability deficits such as diverse chromosome aberrations [33][34][35] or an increased frequency of gene amplification events [36,37], which are thought to be the result of illegitimate DSB repair [38]. Moreover, after irradiation, DNA-PKcs deficient cells accumulate an even higher proportion of chromosome rearrangements [33,39], indicative of a DNA repair defect that mainly results in DSB breaks being rejoined unfaithfully. Thus, the absence of either ATM or DNA-PKcs leads to an abnormal persistence of DSBs and to the accumulation of illegitimate joining events that threaten genomic stability.…”

“…Normal cells repair most of the inflicted damage in the fast repair phase during the very early postirradiation times. The absence of DNA-PKcs kinase always results in a severe delay in the fast repair component (cells derived from SCID mice: [47,48]; M059J cells: [49][50][51]; DNA-PKcs À/À mouse embryonic fibroblasts: [39]). Thus, while normal cells rejoin most of the radio-induced DSBs during the first two hours after irradiation and reach the slow repair phase bearing a low number of DSBs (20-30%), DNA-PKcs-deficient cells reach this phase with a significantly higher number of unrejoined breaks (50-60%) [51].…”