Mechanisms of Rad9 Mediated Checkpoints and Apoptosis

描述(申请人提供)：RAD9介导的检查点和细胞凋亡的机制：细胞周期检查点在维持 通过抑制细胞周期或细胞周期的进展来保持基因组完整性 在DNA受损或不完全的情况下启动细胞程序性死亡 DNA复制。对裂解酵母的研究表明，Rad的成员 检查点蛋白家族包括RAD1、RAD3、RAD9、RAD17、RAD26和 Hus1在DNA损伤和复制的激活中发挥重要作用 检查站。我们已经报道了Rad9可以与Bcl2和Bclxl相互作用 通过位于Rad9蛋白氨基末端的类似BH3的区域， 并能促进哺乳动物细胞的凋亡。DNA损伤增强RAD9 磷酸化并诱导Rad9移动到核膜，在那里它 与Bc l-2共定位。此外，我们的初步数据表明， C-Abl介导的RAD9在Y28上的磷酸化诱导增加的关联 RAD9与BCI-XL有协同作用，并增强RAD9诱导细胞凋亡的作用。 有趣的是，Rad9蛋白被过度磷酸化，某种形式似乎 依赖于细胞周期。此外，我们最近发现Hus1形成了一种 DNA在人皮肤Flow2000成纤维细胞中与增殖细胞核抗原的蛋白质复合体 损坏或复制被禁止。Flow2000细胞对8Gy射线的照射 电离辐射(可诱导G2/M期阻滞，但不会导致细胞凋亡)或羟基尿素 引发了Husi从胞浆到细胞核的移位，在那里它 与增殖细胞核抗原和RAD9共定位。这种核移位和复合体 HUS1与增殖细胞核抗原的形成与细胞周期变化密切相关 对辐射暴露的响应分布。这项提议的目标是 验证Rad9是DNA完整性的重要调节器的假设 确定细胞是否应短暂延迟的检查点路径 DNA损伤后细胞周期进展或死亡及其损伤诱导的复合体 由一组离散的细胞蛋白形成的细胞在 RAD9函数。

DESCRIPTION (PROVIDED BY APPLICANT):MECHANISMS OF RAD9-MEDIATED CHECKPOINTS AND APOPTOSIS: Cell-cycle checkpoints play a critical role in the maintenance of genomic integrity by inhibiting progression through the cell cycle or initiating programmed cell death in the presence of damaged DNA or incomplete DNA replication. Studies in fission yeast implicate that members of the Rad family of checkpoint proteins including Radi, Rad3, Rad9, Radl7, Rad26, and Hus1 play important roles in the activation of DNA damage and replication checkpoints. We have reported that Rad9 can interact with Bcl-2 and Bcl-xL through a BH3-like region located in the amino terminus of the Rad9 protein, and can promote apoptosis in mammalian cells. DNA damage enhances Rad9 phosphorylation and induces Rad9 to move to the nuclear envelope, where it colocalizes with Bcl-2. In addition, our preliminary data indicate that c-Abl-mediated phosphorylation of Rad9 on Y28 induces increased association of Rad9 with BcI-xL and enhances the effect of Rad9 on apoptosis induction. Interestingly, the Rad9 protein is hyperphosphorylated and some form appears to be cell cycle-dependent. Moreover, we have recently found that Hus1 forms a protein complex with PCNA in human skin Flow2000 fibroblasts when DNA is damaged or replication is inhibited. Exposure of Flow2000 cells to 8 Gy ionizing radiation (that induces G2/M-arrest but not apoptosis) or hydroxyurea triggered translocation of Husi from the cytosol to the nucleus, where it colocalized with PCNA and Rad9. This nuclear translocation and the complex formation of Husl with PCNA correlate closely with changes in cell cycle distribution in response to radiation exposure. The goal of this proposal is to test the hypothesis that Rad9 is an important modulator of the DNA integrity checkpoint pathway determining whether a cell should transiently delay cell-cycle progression or die after DNA damage and that damage induced complex formation with a discrete set of cellular proteins plays a critical role in Rad9 function.