Single-Strand Annealing, Conservative Homologous Recombination, Nonhomologous DNA End Joining, and the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by Sparsely or Densely Ionizing Radiation

Abstract

The cell cycle-dependent relative contributions of error-prone single-strand annealing (SSA), error-free conservative homologous recombination (HR), and potentially error-prone nonhomologous DNA end joining (NHEJ) to repair simple (induced by 200 kV X rays) or complex (induced by (241)Am alpha particles) DNA double-strand breaks (DSBs) in Chinese hamster ovary cells are reported for the first time. Cells of the parental cell line AA8 and its derivatives UV41 (SSA-deficient), irs1SF (HR-deficient) and V3 (NHEJ-deficient) were synchronized in G(1) or in S phase, and survival responses after exposure to either type of radiation were measured. It is demonstrated for the first time that in G(1)-phase SSA is negligible for the repair of DSBs of various complexities. HR-deficient cells exposed to X rays or alpha particles in G(1) phase show enhanced radiosensitivity, but this does not necessarily mean that HR is important in G(1) phase. NHEJ appears to be the most important (if not the only) mechanism in G(1) phase acting efficiently on simple DSBs, but complex DSBs are a less preferred target. In contrast to X rays, NHEJ-deficient cells show no cell cycle-dependent variation in sensitivity to alpha particles. Surprisingly, when these cells are exposed to X rays in G(1) phase, they are even more sensitive compared to alpha particles. It is also shown for the first time that in S phase all three mechanisms play a role in the repair of simple and complex DSBs. A defect in SSA confers radiosensitivity to cells in S phase, suggesting that the error-prone SSA mechanism is important for the repair of specific simple and complex DSBs that are not a substrate for HR or NHEJ. The most important mechanism in S phase for the repair of simple and complex DSBs is HR. This is also emphasized by the finding that irs1SF cells, after complementation of their HR defect by human XRCC3 cDNA, show a greater radioresistance than parental cells, whereas resistance to mitomycin C is only partially restored. Complementation confers a greater resistance to alpha particles than X rays, suggesting an important role of HR, especially for the repair of complex DSBs. In S phase, NHEJ is more important than SSA for the repair of simple DSBs, but SSA is more important than NHEJ for the repair of complex DSBs.