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1 December 2008 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
Marlis Frankenberg-Schwager, Manuela Becker, Irmgard Garg, Elke Pralle, Hartmut Wolf, Dieter Frankenberg
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Abstract

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.

Marlis Frankenberg-Schwager, Manuela Becker, Irmgard Garg, Elke Pralle, Hartmut Wolf, and Dieter Frankenberg "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," Radiation Research 170(6), 784-793, (1 December 2008). https://doi.org/10.1667/RR1375.1
Received: 25 February 2008; Accepted: 1 July 2008; Published: 1 December 2008
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