Distinct roles of HR and NHEJ in the repair of ionizing radiation-induced chromosomal double-strand breaks in mammalian cells

DNA double-strand breaks (DSBs) are among the most deleterious lesions induced by ionizing radiation (IR). Unrepaired or misrepaired DSBs can cause genomic instability in the form of chromosome translocations (CTs), leading to cell death or cancer. Classical non-homologous end-joining (c-NHEJ) has been considered the prime DSB repair pathway in mammalian cells. On the other hand, Homologous recombination (HR) has been shown to be crucial for DSB repair in yeast. However, its significance in mammalian cells remained obscured for a long time and started emerging only recently. Alternative end-joining (alt-EJ) acts as a backup pathway to abrogated c-NHEJ or HR, operating with slower kinetics, and is highly error-prone. HR functions strictly in a cell cycle-dependent manner (late S and G2-phase), while c-NHEJ and alt-EJ function throughout the cell cycle. Here, we analyzed the relative contributions of c-NHEJ, HR, and alt-EJ in mammalian cells, which were explicitly irradiated in the G2- phase of the cell cycle and irradiated with low and high doses of IR. Such G2-specific analysis facilitated the simultaneous investigation of all DSB repair pathways at varying degrees of DSB loads. Our results suggest that HR is indispensable for repairing chromosomal DSBs at physiologically relevant low IR doses (≤1Gy). In contrast, a shift towards c-NHEJ is evident when cells are exposed to high doses of IR. Both c-NHEJ and HR suppress CT formation, which strongly depends on PARP- 1 mediated alt-EJ.Furthermore, cells opt to actively suppress c-NHEJ at physiologically relevant IR doses and HR with increasing DSB loads at high doses of IR. Our results highlight the importance of HR in repairing chromosomal DSBs in mammalian cells exposed to physiologically relevant IR doses. The underlying molecular mechanisms behind such DSB load-dependent repair pathway switch are currently under investigation. (author)