College of Veterinary Medicine, Department of Microbiology & Molecular Genetics, Department of Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA.
College of Veterinary Medicine, Department of Microbiology & Molecular Genetics, Department of Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA.
DNA Repair (Amst). 2020 Oct;94:102925. doi: 10.1016/j.dnarep.2020.102925. Epub 2020 Jul 8.
It has recently been established that the marked sensitivity of ATM deficient cells to topoisomerase poisons like camptothecin (Cpt) results from unrestrained end-joining of DNA ends at collapsed replication forks that is mediated by the non-homologous end joining [NHEJ] pathway and results in the induction of copious numbers of genomic alterations, termed "toxic NHEJ". Ablation of core components of the NHEJ pathway reverses the Cpt sensitivity of ATM deficient cells, but inhibition of DNA-PKcs does not. Here, we show that complete ablation of DNA-PKcs partially reverses the Cpt sensitivity of ATM deficient cells; thus, ATM deficient cells lacking DNA-PKcs are more resistant to Cpt than cells expressing DNA-PKcs. However, the relative sensitivity of DNA-PKcs proficient ATM deficient cells is inversely proportional to DNA-PKcs expression levels. These data suggest that DNA-PK may phosphorylate an ATM target (that contributes to Cpt resistance), explaining partial rescue of Cpt sensitivity in cells expressing high levels of DNA-PKcs. Although crippling NHEJ function by mutagenic blockade of the critical ABCDE autophosphorylation sites in DNA-PKcs also sensitizes cells to Cpt, this sensitization apparently occurs by a distinct mechanism from ATM ablation because blockade of these sites actually rescues ATM deficient cells from toxic NHEJ. These data are consistent with autophosphorylation of the ABCDE sites (and not ATM mediated phosphorylation) in response to Cpt-induced damage. In contrast, blockade of S3205 (an ATM dependent phosphorylation site in DNA-PKcs) that minimally impacts NHEJ, increases Cpt sensitivity. In sum, these data suggest that ATM and DNA-PK cooperate to facilitate Cpt-induced DNA damage, and that ATM phosphorylation of S3205 facilitates appropriate repair at collapsed replication forks.
最近已经证实，ATM 缺陷细胞对拓扑异构酶抑制剂如喜树碱（Cpt）的敏感性显著增加，这是由于复制叉崩溃时未受控制的 DNA 末端连接，这种连接是由非同源末端连接[NHEJ]途径介导的，导致大量基因组改变的诱导，称为“毒性 NHEJ”。NHEJ 途径的核心成分缺失可逆转 ATM 缺陷细胞对 Cpt 的敏感性，但 DNA-PKcs 的抑制却不能。在这里，我们发现 DNA-PKcs 的完全缺失部分逆转了 ATM 缺陷细胞对 Cpt 的敏感性；因此，缺乏 DNA-PKcs 的 ATM 缺陷细胞比表达 DNA-PKcs 的细胞对 Cpt 的耐药性更强。然而，DNA-PKcs 功能正常的 ATM 缺陷细胞的相对敏感性与 DNA-PKcs 的表达水平成反比。这些数据表明，DNA-PK 可能磷酸化 ATM 靶标（有助于 Cpt 耐药性），解释了表达高水平 DNA-PKcs 的细胞中 Cpt 敏感性的部分恢复。尽管通过 DNA-PKcs 关键 ABCDE 自动磷酸化位点的突变阻断严重削弱 NHEJ 功能也会使细胞对 Cpt 敏感，但这种敏化显然是通过与 ATM 缺失不同的机制发生的，因为这些位点的阻断实际上使 ATM 缺陷细胞从毒性 NHEJ 中拯救出来。这些数据与 ABCDE 位点的自动磷酸化（而不是 ATM 介导的磷酸化）对 Cpt 诱导的损伤的反应一致。相反，S3205 （DNA-PKcs 中的一个 ATM 依赖性磷酸化位点）的阻断（对 NHEJ 的影响最小）会增加 Cpt 的敏感性。总之，这些数据表明，ATM 和 DNA-PK 合作促进 Cpt 诱导的 DNA 损伤，而 ATM 对 S3205 的磷酸化促进了复制叉崩溃时的适当修复。
