FIG 3.
Proteasome activities are essential for RAD6-mediated HR and NHEJ repair. (A) Diagrams of the HR and NHEJ reporter systems. Detailed descriptions are provided in Materials and Methods. Pcmv, cytomegalovirus promoter; SV40, simian virus 40. G and FP indicate the separated parts of the GFP gene. (B) Elevated levels of HR and NHEJ repair regulated by RAD6 depend on the presence of proteasome activities. Cells carrying the HR or NHEJ reporters were transfected with or without Myc-tagged RAD6A, and cells were treated with 25 μM MG132 for another 8 h or not treated, as indicated. The HR and NHEJ repair efficiencies were calculated using the specific reporters as previously described (12). The error bars indicate the standard deviations from three biological replicates. (C) RAD6 overexpression promotes DNA damage repair, while this accelerating effect was abolished by the inhibition of proteasome activity. HEK293T cells transfected with an empty vector expressing GFP as a control or GFP-tagged RAD6A were treated with 25 μM MG132 for 8 h or not treated, as indicated. Cells were then subjected to X-ray irradiation at a dosage of 80 kV for 5 min and recovered at the indicated times. Lastly, cells were harvested and lysed for Western blot analyses with the indicated antibodies. (D) RAD6 promotes the upregulation of proteasome activities during DNA damage repair, while knockdown of RAD6 expression abolishes the proteasome activity increase during DNA damage repair. Control HEK293T cells and cells transfected with Myc-tagged RAD6A or RAD6A/B-specific siRNAs were subjected to X-ray irradiation as described above. Cells were harvested at specific recovery times, and total cell extracts were prepared. The proteasome activities were examined using the Amplit fluorimetric proteasome 20S activity assay kit (AAT Bioquest, CA; catalog number 13456) according to the manufacturer’s protocol. The error bars indicate the standard deviations from three biological replicates. **, P < 0.05. (E) X-ray irradiation induces an increase in nuclear proteasome activities, depending on the presence of RAD6. HEK293T cells were treated with X-ray irradiation as described above. Nuclear (Nu) and cytoplasmic (Cyto) fractions were prepared using a nuclear and cytoplasmic protein extraction kit (product number P0028; Beyotime, Jiangsu Province, People's Republic of China) according to the manufacturer's instruction. Then, the proteasome activities of each fraction were examined. The error bars indicate the standard deviations from three biological replicates. Cont, control.