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Rad9、Rad1和Hus1组成的检查点蛋白复合体9-1-1在细胞周期调控中的作用。这些报道表明，9-1-1复合体可能在检测G2/M期各种DNA损伤方面发挥作用。已有研究表明，9-1-1复合体被各种DNA损伤上的酶所磷酸化，其组成在结构上类似于增殖细胞核抗原同源三聚体。通过这些研究，检查点机制的框架基本揭开了面纱，但仍然存在一些问题，如9-1-1复合体具有哪些独特和必要的功能，9-1-1复合体如何作为DNA损伤传感器发挥重要作用。本研究旨在通过建立9-1-1复合体的条件基因敲除细胞系来阐明9-1-1复合体的独特和重要作用。建立了RAD1条件基因敲除细胞系和HUS1条件基因敲除细胞系，而RAD9条件基因敲除细胞系未能建立。因此，利用重组microRNA对RAD9信使RNA进行了敲除。RAD1基因敲除后，细胞死亡，而Hus1基因敲除后，细胞存活。为了评估已报道的RAD1外核酸酶活性在细胞周期调控中的重要性，构建了RAD1序列中保守的潜在外核酸酶基序的点突变体，并生产了相应的重组RAD1蛋白用于外核酸酶活性的检测。然而，野生型Rad1及其突变体没有观察到任何外源核酸酶活性。观察到Hus1在条件基因敲除细胞系中普遍存在，这与先前的报道一致。观察到Hus1泛素化持续发生，且泛素化程度不受多种抗癌药物作用的影响。

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.