|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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.