The MRAD9 Radioresistance Gene

描述(申请人提供)：生物对辐射暴露的反应方式很重要，因为诱发的DNA损伤可能导致死亡、突变或癌症。之前的工作主要集中在裂解酵母S.pombe rad9上，这是一种促进抗伽玛射线、抗紫外线、抗DNA复制抑制剂羟基脲的基因，并调节相关的细胞周期检查点。我们鉴定了人(HRAD9)和小鼠(Mrad9)的同源基因，并发现相应的cDNA部分弥补了rad9：：ura4+酵母所显示的几个缺陷。此外，我们还发现，HRAD9蛋白在其C-末端与检查点蛋白HHUS1和HRAD1结合，在其N-末端含有一个BH3样结构域，可与抗凋亡蛋白bcl2和bclxl结合，当过表达时可引起细胞凋亡。我们还发现，这种多功能蛋白可以与p53结合，共同调节p21。这项建议的主要重点是建立并扩展我们使用酵母和哺乳动物系统积累的大量数据。具体地说，我们将利用在前一个资助期构建的Mrad9基因敲除细胞来解决旨在阐明Mrad9功能的明确假设，从而解释细胞对DNA损伤的反应的分子机制。这些假说包括：1)基因突变导致对DNA损伤的敏感性，至少部分原因是碱基切除修复的缺陷，而不仅仅是细胞周期检查点；2)Mrad9在有或没有外源DNA损伤剂的情况下调节基因组的稳定性，它是通过重组蛋白的作用而调节的；3)新发现的结构和功能相关的RAD9B(HRAD9B)，也在介导对DNA损伤的抗性和维持基因组稳定方面发挥重要作用，并且两个相关蛋白的活性是部分冗余的。实验方法将通过Mrad9突变细胞的分子、细胞和生化特征来解决这些假说，并且Mrad9B Paralog中的细胞也发生了改变。这些研究将从分子到细胞水平研究Mrad9的功能，确定Mrad9活性的结构基础，并可能影响放射治疗以及了解DNA损伤的遗传易感性。

DESCRIPTION (provided by applicant): The way organisms respond to radiation exposure is important since induced DNA lesions 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, and regulates the associated cell cycle checkpoints. We identified human (HRAD9) and mouse (Mrad9) orthologues, and the corresponding 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 by us using yeast and mammalian systems. Specifically, we will make use of Mrad9 knockout cells constructed during the previous funding period to address well-defined hypotheses designed to elucidate Mrad9 function, and thus explain molecular mechanisms involved in the cellular response to DNA damage. These hypotheses include: 1) Mutations in the gene cause sensitivity to DNA damage at least in part because of defects in base excision repair, not just in cell cycle checkpoints; 2) Mrad9 regulates genomic stability in the presence or absence of exogenous DNA damaging agents, and it is mediated through the action of recombination proteins; 3) A newly discovered structurally and functionally related paralog of Rad9, called Mrad9B (HRAD9B), also plays important roles in mediating resistance to DNA damage and maintaining genomic stability, and the activities of the two related proteins are partially redundant. Experimental approaches will address these hypotheses through the molecular, cellular and biochemical characterization of Mrad9 mutant cells, and cells also altered in the Mrad9B paralog. These studies will examine Mrad9 function from molecular to cellular levels, define the structural basis for Mrad9 activity, and could impact on radiotherapy as well as on understanding genetic susceptibility to DNA damage.