Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle

Abstract

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of γ-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G₁, greater impairment in S, and a substantial defect in late S/G₂. Furthermore, the radiosensitivity of irs1SF cells is slight in G₁ but dramatically higher in late S/G₂, while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G₂, where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.

ACKNOWLEDGMENTS

We thank P. Jeggo for kindly providing AA8, V3, and DNA-PK_cs-complemented V3 cells and R. Greinert for providing K1 cells; xrs-6 cells (European Collection of Cell Cultures) are commercially available.

Financial support was provided by the Deutsche Forschungsgemeinschaft (grants Lo 677/1-1 and Lo 677/1-2), the Radiation Protection Programme of the European Community (grant FIGH-CT-1999-00012), and the Bundesministerium für Bildung und Forschung via the Forschungszentrum Karlsruhe (grant 02S8132). A portion of this work was prepared under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract no. W-7405-ENG-48 and was funded by the Low-Dose Radiation Research Program, Biological and Environmental Research, U.S. Department of Energy (grant SCW0389/0008).