Mrad9 Radioresistance Gene

生物体对辐射暴露的反应方式很重要， 诱导的DNA损伤错误修复或未修复可导致死亡，突变 或者癌症以前的努力集中在裂变酵母S。pombe rad 9基因 增强了对伽马射线的抵抗力，紫外线的抵抗力， 复制抑制剂羟基脲（flU）和早期S期以及G2 检查点控制最近，人（HRAD 9）和小鼠（Mrad 9）的基因组和cDNA 已经鉴定了直向同源物，并且发现cDNA部分 补充由RAD 9：：URA 4+酵母证明的几个缺陷。而且我们 发现HRAD 9蛋白结合检查点蛋白HHUS 1和HRAD 1， C-末端区域，并且在其N-末端区域含有BH 3样结构域， 可结合抗凋亡蛋白BCL-2和BCL-xL，并可引起凋亡 当过度表达时。我们还发现这种多功能蛋白质可以结合 p53和共调节p21。本提案的主要重点是建立在 大量的数据积累与酵母和哺乳动物系统，并集中在 Mrad 9在哺乳动物中的作用具体而言，这项建议将利用 Mrad 9敲除细胞和小鼠来解决这一假设，就像酵母 Mrad 9基因在细胞对DNA损伤的反应中起关键作用， 抑制DNA复制。至少部分解释RAD 9的工作模型 在细胞周期进展和/或凋亡中起作用，并且其方面将 以下是需要解决的问题： 在DNA损伤后，ATM介导RAD 9（和p53在丝氨酸-15上）的磷酸化， 调节p21水平;增加p21丰度抑制Rb磷酸化， 其处于不能激活E2 F1的形式，因此转录多个 参与细胞周期进程、凋亡和相关过程的基因是 不诱导。实验目的：1）构建Mrad 9基因敲除细胞和小鼠; 2)在细胞水平上表征Mrad 9突变的生物学影响， 包括对DNA损伤剂的敏感性、检查点控制和细胞凋亡; 3)通过检查Mrad 9突变的变化来表征Mrad 9突变的分子影响， 蛋白质在工作模型中的地位; 4）解决上位性 Mrad 9与ATM、p2]和p53在生物学和 分子影响; 5）进行结构/功能研究以确定Mrad 9区域 重要的活性; 6）评估Mrad 9在肿瘤发生中的作用;和7） 后续的初步结果表明，Mrad 9参与胚胎发育， 发育或器官维持/发育及其与 蛋白质对DNA损伤的反应。这些研究将考察 从分子到细胞水平，以及整个动物中的Mrad 9， 对放射治疗的影响及其作为遗传标记的潜力 表明对DNA损伤的敏感性。

The way organisms respond to radiation exposure is important since induced DNA lesions misrepaired or left unrepaired can lead to death, mutation or cancer. Previous efforts have focused on fission yeast S. pombe rad9, a gene that promotes gamma-ray resistance, UV-resistance, resistance to the DNA replication inhibitor hydroxyurea (flU), and early S phase as well as G2 checkpoint control. Recently, human (HRAD9) and mouse (Mrad9) genomic and cDNA orthologues have been identified and the cDNAs were found to partially complement several defects demonstrated by rad9::ura4+ yeast. Furthermore, we found that HRAD9 protein binds the checkpoint proteins HHUS1 and HRAD1 at its C-terminal region, and contains a BH3-like domain at its N-terminal region that can bind the anti-apoptotic proteins BCL-2 and BCL-xL, and can cause apoptosis when overexpressed. We also found that this multifunctional protein can bind p53 and co-regulate p21. The major focus of this proposal builds on and extends a large amount of data accrued with yeast and mammalian systems, and centers on the function of Mrad9 in mammals. Specifically, this proposal will make use of Mrad9knockout cells and mice to address the hypothesis that, like the yeast gene, Mrad9plays a key role in the response of cells to DNA damage or inhibition of DNA replication. A working model to at least partly explain RAD9 function in cell cycle progression and/or apoptosis, and aspects of which will be addressed, is the following: after DNA damage, ATM mediates phosphorylation of RAD9 (and p53 on serine-15), that regulates p21 levels; increased p21 abundance inhibits Rb phosphorylation, which is in a form unable to activate E2F1, and thus transcription of multiple genes involved in cell cycle progression, apoptosis and related processes are not induced. Experimental goals are 1) construct Mrad9knockout cells and mice; 2) characterize the biological impact of Mrad9mutations at the cellular level, including sensitivity to DNA damaging agents, checkpoint control and apoptosis; 3) characterize the molecular impact of Mrad9 mutations by examining changes in the status of proteins in the working model; 4) address the epistatic relationship between Mrad9 and ATM, p2] and p53 in terms of biological and molecular impact; 5) perform structure/function studies to define Mrad9regions important for activity; 6) assess the role of Mrad9in tumorigenesis; and 7) follow up preliminary results indicating that Mrad9 participates in embryonic development, or organ maintenance/development and its relation to the role of the protein in response to DNA damage. These studies will examine the function of Mrad9 from molecular to cellular levels, and in whole animals, and could impact on radiotherapy and on understanding its potential as a genetic marker indicative of susceptibility to DNA damage.