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DNA double-strand breaks (DSBs) are usually repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). NHEJ is thought to be the predominant pathway operating in mammalian cells functioning in all phases of the cell cycle, while HR works in the late-S and G2 phases. However, relative contribution, competition, and dependence on cell cycle phases are not fully understood. We previously developed a system to trace the fate of DSBs in the human genome by introducing the homing endonuclease I-SceI site into the thymidine kinase (TK) gene of human lymphoblastoid TK6 cells. Here, we use this system to investigate the relative contribution of HR and NHEJ for repairing I-SceI-induced DSBs under various conditions. We used a novel transfection system, Amaxa nucleofector, which directly introduces the I-SceI expression vector into cell nuclei. Approximately 65% of transfected cells expressed the I-SceI enzyme and over 50% of the cells produced a single DSB in the genome. The relative contribution of NHEJ and HR for repairing the DSB was approximately 100:1 and did not change with transfection efficiency. Cotransfection with KU80-siRNA significantly diminished KU80 protein levels and decreased NHEJ activity, but did not increase HR. We also investigated HR and NHEJ in synchronized cells. The HR frequency was 2-3 times higher in late-S/G2 phases than in G1, whereas NHEJ was unaffected. Even in late-S/G2 phases, NHEJ remained elevated relative to HR. Therefore, NHEJ is the major pathway for repairing endonuclease-induced DSBs in mammalian cells even in late-S/G2 phase, and does not compete with HR.

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