高质量抗体揭示Rad1蛋白在细胞中新的潜在活动机理

一组在进化上(从酵母到人)保守的基因Rad9、Rad1和Hus1在细胞周期监控点调控和DNA损伤修复中发挥重要作用．这三个蛋白可以形成环形异源三聚体，即9-1-1蛋白复合体．9-1-1复合体被认为是Rad9、Rad1和Hus1行使功能的主要形式．到目前为止，没有一个好的抗Rad1的抗体，严重阻碍了对Rad1和9-1-1复合体的研究．在本研究中，我们成功地制备了一株小鼠抗Rad1蛋白的单克隆抗体．这个抗体能够有效地检测小鼠和人的内源Rad1蛋白，可以用于酶联免疫吸附、蛋白质免疫印迹、免疫共沉淀和免疫荧光等实验．利用该抗体，我们发现在DNA损伤剂羟基脲(HU)的诱导下，小鼠Rad1蛋白在Rad9^+/+小鼠胚胎干细胞中表达明显增加，而在Rad9^-/-的小鼠胚胎干细胞中没有观察到该现象，这表明Rad9对Rad1的蛋白表达有调控作用．此外，内源的Rad1蛋白主要分布在细胞质中，在HU处理后并没有迁移进入细胞核的现象，这与先前广泛被人们所接受的在DNA损伤压力下Rad1和Hus1能够迁移进入细胞核并与Rad9形成9-1-1蛋白复合体的说法相矛盾．综合看来，Rad1和9-1-1蛋白复合体的分子作用机制比预期的要复杂，我们成功制备的Rad1单克隆抗体将成为研究Rad1以及9-1-1蛋白复合体的强有力的工具．

Novel Cellular Activities of The Cell Cycle Checkpoint Protein Rad1 Revealed by a New High-quality anti-Rad1 Antibody

Rad9, Rad1 and Hus1 are critical for the cell cycle checkpoint and can form a heterotrimer complex called 9-1-1 complex which was supposed to play important roles in the cell cycle checkpoint and other activities required for the maintenance of genome integrity. However, lack of high quality anti-Rad1 antibodies has seriously hindered the research on Rad1 as well as working mechanisms of the 9-1-1 complex at molecular level. In this study, a mouse anti-Rad1 monoclonal antibody (mAb) was successfully generated. The mAb possesses high affinity and specificity, and recognizes both endogenous mouse Rad1 (mRad1) and human Rad1 (hRad1) proteins and was successfully used in ELISA, Western blot analysis, immunoprecipitation and immunofluorescence assays. Using this mAb, we found that mRad1 protein expression was increased in Rad9^+/+ mouse embryonic stem (MES) cells after hydroxyurea (HU, a genotoxic agent) treatment while not in Rad9^-/- MES cells, suggesting that mRad1 expression is under Rad9 regulation. Furthermore, endogenous mRad1 was distributed mainly in the cytoplasm and did not migrate to the nucleus after HU treatment, contradicting the generally accepted hypothesis that Rad9, Rad1 and Hus1 form the 9-1-1 complex in the nucleus in response to genotoxic stresses. In summary, the exact molecular roles of Rad1 and the 9-1-1 complex are likely more complicated than previously expected and this anti-Rad1 mAb is a powerful tool for the future investigation on Rad1 as well as the 9-1-1 complex.