JOURNAL ARTICLE

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 H(2)O(2) in mammalian cells

Marlis Frankenberg-Schwager, Manuela Becker, Irmgard Garg, Elke Pralle, Hartmut Wolf, Dieter Frankenberg
Radiation Research 2008, 170 (6): 784-93
19138034
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 H(2)O(2)-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 H(2)O(2) in G(1) or S phase of the cell cycle and their colony-forming ability was assayed. In G(1) phase, NHEJ was the most important-if not the only-mechanism to repair H(2)O(2)-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 H(2)O(2) relative to parental AA8 cells in G(1) 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 H(2)O(2)-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 G(1) phase showed an increased sensitivity to high concentrations of H(2)O(2) relative to the parental wild-type cells and to HR-deficient cells, which may be interpreted in terms of a specific type of H(2)O(2)-induced damage requiring SSA for repair after its transfer into S phase. In S phase, HR is the most important mechanism to repair H(2)O(2)-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 H(2)O(2) 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 H(2)O(2) in S phase compared to G(1) phase. This effect is likely due to the fact that topoisomerase II, which is expressed only in proliferating cells, is a target of H(2)O(2), resulting in enhanced accumulation of DSBs and killing of cells treated in S phase with H(2)O(2). The relative sensitivities to H(2)O(2) differ by orders of magnitude for the four cell lines. This seems to be caused mainly by H(2)O(2)-mediated poisoning of topoisomerase IIalpha rather than by a defect in DSB repair.

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