Analysis of roles of checkpoint protein complex 9-1-1 on cell cycle regulation

有越来越多的报道描述了由Rad 9、Rad 1和Hus 1组成的检查点蛋白复合物9-1-1在细胞周期调控中的作用。这些研究表明，9-1-1复合物可能在G2/M期对多种DNA损伤具有敏感作用。有报道9-1-1复合物在各种DNA损伤时被激酶磷酸化，其组成与PCNA同源三聚体结构相似。通过这些研究，检验点机制的框架已基本被揭示，但仍存在一些问题，如9-1-1复合物的独特功能和重要功能，以及9-1-1复合物作为DNA损伤传感器的重要作用。本研究利用胚胎癌细胞系F9系统建立条件性基因敲除细胞系，旨在阐明9-1-1复合物的独特和重要作用。建立了Rad 1和Hus 1条件性基因敲除细胞系，但Rad 9条件性基因敲除细胞系未能建立。因此，重组微RNA用于敲低Rad 9信使RNA。通过敲除Rad 1基因，观察到细胞走向死亡，而通过敲除Hus 1基因，观察到细胞存活。为了评估Rad 1的外切核酸酶活性对细胞周期调控的重要性，构建了Rad 1序列中保守的潜在外切核酸酶基序的点突变体，并制备了相应的重组Rad 1蛋白，用于外切核酸酶测定。然而，未观察到野生型Rad 1及其突变体的任何外切核酸酶活性。Hus 1在条件敲除细胞系中被泛素化的观察结果与先前的报道一致。并且观察到Hus 1的泛素化持续发生，并且泛素化的程度不因各种抗癌药物的治疗而改变。

There are accumulating reports describing roles of checkpoint protein complex 9-1-1 consisting of Rad9, Rad1 and Hus1 on cell cycle regulations. By those reports it is suggested that 9-1-1 complex may work for sensing various kinds of DNA damages at G2/M phase. It has been reported that 9-1-1 complex is phosphorylated by kinases on various DNA damages, and that composition of 9-1-1 complex is structurally resemble to PCNA homo trimer. Through these researches, framework of checkpoint mechanism is almost unveilings, however, there are still questions, such as what functions are unique and essential for 9-1-1 complex, and how 9-1-1 complex play an important role as a DNA damage sensor. This study aimed to explain unique and essential roles of 9-1-1 complex by establishing conditional knock out cell line using embrional carcinoma cell line F9 system. Rad1 conditional knock out cell line and Hus1 conditional knock out cell line were established, however, Rad9 conditional knock out cell line was not able to do. Therefore, recombinant micro RNA was utilized for knock down of Rad9 messenger RNA. By knock out of Rad1 gene, the cell going to death was observed, in contrast, knock out of Hus1 gene, the cell was alive. To assess the importance of reported exo-nuclease activity of Rad1 on cell cycle regulation, point mutants of conserved potential exo-nuclease motif in Rad1 sequence were constructed and corresponding recombinant Rad1 proteins were produced and utilized for exo-nuclease assay. However any exo-nuclease activity of wild type Rad1 and its mutants were not observed. The observation that Hus1 was ubiquinated in conditional knock out cell line was consistent with the previous report. And it was observed that the ubiquitination of Hus1 was constantly occurred, and that the extent of the ubiquitination was not altered by the treatment with various anticancer drugs.