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

¹ Department of Cell Biology, Physiology, and Immunology, Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Spain; ² Molecular Oncology Program, Spanish National Cancer Centre, Madrid, Spain; ³ Department of Microbiology, Boston University School of Medicine, Boston, Massachussetts; and ⁴ Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen, Germany

Requests for reprints: Anna Genescà, Cell Biology Unit, Ed CS, Universitat Autònoma de Barcelona, Bellaterra, Spain. Phone: 34-93-581-1498; Fax: 34-93-581-2295; E-mail: anna.genesca{at}uab.es.

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, DNA-PKcs^–/– 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^–/– 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^–/– MEFs than in their wild-type counterparts. Spectral karyotyping-fluorescence 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^–/– MEFs. Because DNA-PKcs^–/– MEFs show a strong G₁ checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs^–/– 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.

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