Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA, School of Biomedical Sciences, Kent State University, Kent, OH 44234, USA, Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Nucleic Acids Res. 2013 Dec;41(22):10157-69. doi: 10.1093/nar/gkt812. Epub 2013 Sep 9.
Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.
暴露于遗传毒性剂，如电离辐射（IR），会产生双链断裂，哺乳动物细胞主要通过非同源末端连接（NHEJ）修复。Ku70 被鉴定为蛋白水解细胞周期蛋白 E（CycE）片段 p18CycE 的相互作用伙伴。p18CycE 在白血病细胞的 IR 诱导凋亡过程中内源性产生，在对 IR 敏感的上皮肿瘤细胞中稳定表达。γH2AX IR 诱导焦点（IRIFs）和彗星试验表明，p18CycE 表达细胞中的 NHEJ DNA 修复无效。DNA 下拉和染色质募集试验表明，NHEJ 因子对双链断裂的保留，但不是募集，减少了。同样，p18CycE 表达细胞中磷酸化 T2609 和 S2056-DNA-PKcs 的 IRIFs 及其靶标 S1778-53BP1 大大减少。因此，DNA-PKcs 染色质关联也增加了。当在白血病细胞和稳定表达 p18CycE 的上皮细胞中产生 p18CycE 时，53BP1 IRIFs 受到抑制。共济失调毛细血管扩张突变被激活，但不是其 53BP1 和 MDC1 靶标。这些数据表明，p18CycE 通过干扰 DNA-PKcs 构象和/或二聚化对 NHEJ 产生深远影响，这是有效 DNA 修复所必需的，使 p18CycE 表达细胞对 IR 更敏感。这些研究为人类肿瘤细胞中 NHEJ 失调的提供了独特的机制见解，其中 NHEJ 核心成分的缺陷很少见。
