Marangoni E, Foray N, O'Driscoll M, Douc-Rasy S, Bernier J, Bourhis J, Jeggo P
Unité Propre de l'Enseignement Supérieur 'Radiosensibilité-Radiocarcinogenèse Humaine' (UPRES EA no. 2710, Pr. Eschwege), IFR no. 54, Institut Gustave Roussy, Villejuif, France.
Nucleic Acids Res. 2000 Dec 1;28(23):4778-82. doi: 10.1093/nar/28.23.4778.
DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.
DNA非同源末端连接是哺乳动物细胞中DNA双链断裂（DSB）修复的主要机制，该过程需要DNA依赖蛋白激酶（DNA-PK），它是一个由460 kDa的大型催化亚基（DNA-PKcs）和与双链DNA末端结合的异源二聚体Ku70-Ku80组成的复合体。DNA-PK三个亚基中任何一个发生突变都会导致极端的放射敏感性和DSB修复缺陷。在此我们表明，导入中国仓鼠卵巢细胞系CHO-K1的Ku80的283个C末端氨基酸具有显性负效应。通过Northern印迹分析验证了Ku(449-732)在CHO细胞中的表达，其导致Ku依赖的DNA末端结合活性降低，通过脉冲场凝胶电泳测定的DSB修复能力减弱，以及通过克隆形成存活测定的放射抗性降低。在分子和细胞水平观察到的稳定变化表明，Ku80的这一片段赋予显性负效应，为使放射抗性细胞敏感化提供了重要机制。
