Frankenberg-Schwager, M., Becker, M., Garg, I., Pralle, E., Wolf, H. and Frankenberg, D., The Role of Nonhomologous DNA End Joining, Conservative Homologous Recombination, and Single-Strand Annealing in the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by H₂O₂ in Mammalian Cells. Radiat. Res. 170, 784–793 (2008).

The purpose of this study was to investigate the cell cycle-dependent role of nonhomologous DNA end joining (NHEJ), conservative homologous recombination (HR), and single-strand annealing (SSA) for the repair of simple DNA double-strand breaks (DSBs) induced by H₂O₂-mediated OH radicals in CHO cells. Cells of the cell lines V3 (NHEJ-deficient), irs1SF (HR-deficient) and UV41 (SSA-deficient) and their parental cell line AA8 were exposed to various concentrations of H₂O₂ in G₁ or S phase of the cell cycle and their colony-forming ability was assayed. In G₁ phase, NHEJ was the most important—if not the only—mechanism to repair H₂O₂-mediated DSBs; this was similar to results obtained in a parallel study of more complex DSBs induced by sparsely or densely ionizing radiation. Unlike HR (irs1SF)- and SSA (UV41)-deficient cells, the sensitivity of NHEJ-deficient V3 cells to H₂O₂ relative to parental AA8 cells in G₁ phase is about 50 times higher compared to 200 kV X rays. This points to a specific role of the catalytic subunit of DNA-PK for efficient NHEJ of H₂O₂-mediated DSBs that are located at sites critical for the maintenance of the higher-order structure of cellular DNA, whereas X-ray-induced DSBs are distributed stochastically. Surprisingly, SSA-deficient cells in G₁ phase showed an increased sensitivity to high concentrations of H₂O₂ relative to the parental wild-type cells and to HR-deficient cells, which may be interpreted in terms of a specific type of H₂O₂-induced damage requiring SSA for repair after its transfer into S phase. In S phase, HR is the most important mechanism to repair H₂O₂-mediated DSBs, followed by NHEJ. In contrast, the action of error-prone SSA may not be beneficial, since SSA-deficient cells are three times more resistant to H₂O₂ than wild-type AA8 cells. This is likely due to channeling of DSBs into the error-free HR repair pathway or into the potentially error-prone NHEJ pathway. Cells with or without a defect in DSB repair are considerably more sensitive to H₂O₂ in S phase compared to G₁ phase. This effect is likely due to the fact that topoisomerase II, which is expressed only in proliferating cells, is a target of H₂O₂, resulting in enhanced accumulation of DSBs and killing of cells treated in S phase with H₂O₂. The relative sensitivities to H₂O₂ differ by orders of magnitude for the four cell lines. This seems to be caused mainly by H₂O₂-mediated poisoning of topoisomerase IIα rather than by a defect in DSB repair.