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 homolog ous recombination (HR). Inactivation of NHEJ is known to lead to an elevate d level of spontaneous and radiation-induced chromosomal rearrangements ass ociated with an increased risk of tumorigenesis, This has raised the idea o f a caretaker role for NHEJ, It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influe nce of a defect in NHEJ on the formation of genomic rearrangements, we appl ied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing r adiation, After 80 GY of X-irradiation at a high dose rate (23 Gy/min), wil d-type cells repaired 50% of the induced DSBs within 23 h by incorrect rejo ining. This frequency of DSB misrejoining is considerably reduced in NHEJ-d eficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 GS over a period of 14 days under repair conditions, led to no detect able 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 cel ls form genomic rearrangements frequently. These: repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in R AD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its c aretaker role, also the potential to effect genomic rearrangements. We prop ose 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.