Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks.
Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis. This has raised the idea of a caretaker role for NHEJ. It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation. After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining. This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells. This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies. However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently. These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements. We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.
Rothkamm
K
K
Fachrichtung Biophysik, Universität des Saarlandes, D-66421 Homburg/Saar, Germany.
Kühne
M
M
Jeggo
P A
PA
Löbrich
M
M
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004265:DNA Helicases; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects*; D045643:DNA Repair Enzymes; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005347:Fibroblasts; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D016193:G1 Phase / Q000502:physiology; D015321:Gene Rearrangement / Q000528:radiation effects; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; C000607891:DNL4 protein, S cerevisiae; D005656:Fungal Proteins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP