Postreplicative joining of DNA double-strand breaks causes genomic instability in DNA-PKcs-deficient mouse embryonic fibroblasts.
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.
Martín
Marta
M
Department of Cell Biology, Physiology, and Immunology, Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Genescà
Anna
A
Latre
Laura
L
Jaco
Isabel
I
Taccioli
Guillermo E
GE
Egozcue
Josep
J
Blasco
María A
MA
Iliakis
George
G
Tusell
Laura
L
D000818:Animals; D002478:Cells, Cultured; D002869:Chromosome Aberrations / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase / Q000172:deficiency*; D004622:Embryo, Mammalian; D005260:Female; D005347:Fibroblasts / Q000201:enzymology* / Q000502:physiology / Q000528:radiation effects / Q000648:ultrastructure*; D042822:Genomic Instability / Q000502:physiology* / Q000528:radiation effects; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D011247:Pregnancy; D012854:Sister Chromatid Exchange; D016615:Telomere / Q000502:physiology
D051747:DNA-Activated Protein Kinase