The MRAD9 Radioresistance Gene

描述（由申请人提供）：生物体对辐射照射的反应方式很重要，因为诱导的DNA损伤可导致死亡、突变或癌症。以前的努力集中在裂变酵母S。pombe rad 9是一种促进γ射线抗性、UV抗性、对DNA复制抑制剂羟基脲的抗性并调节相关细胞周期检查点的基因。我们鉴定了人（HRAD 9）和小鼠（Mrad 9）的直向同源物，并且发现相应的cDNA部分地补充了由rad 9：：ura 4+酵母证明的几个缺陷。此外，我们发现HRAD 9蛋白在其C-末端区域结合检查点蛋白HHUS 1和HRAD 1，并且在其N-末端区域包含BH 3样结构域，其可以结合抗凋亡蛋白BCL-2和BCL-xL，并且当过表达时可以引起凋亡。我们还发现，这种多功能蛋白可以结合p53并共同调节p21。这项提案的主要重点是建立在我们使用酵母和哺乳动物系统积累的大量数据的基础上，并扩展了这些数据。具体来说，我们将利用在前一个资助期内构建的Mrad 9敲除细胞来解决旨在阐明Mrad 9功能的明确假设，从而解释细胞对DNA损伤反应的分子机制。这些假设包括：1）基因突变引起对DNA损伤的敏感性，至少部分是因为碱基切除修复的缺陷，而不仅仅是细胞周期检查点的缺陷; 2）Mrad 9在存在或不存在外源DNA损伤剂的情况下调节基因组稳定性，并且其通过重组蛋白的作用介导; 3）一种新发现的结构和功能上与Rad 9相关的蛋白质，称为Mrad 9 B（HRAD 9 B），在介导抗DNA损伤和维持基因组稳定性方面也起着重要作用，两个相关蛋白的活性部分冗余。实验方法将通过Mrad 9突变细胞的分子、细胞和生物化学表征来解决这些假设，并且细胞也在Mrad 9 B parabolism中改变。这些研究将从分子到细胞水平检查Mrad 9的功能，确定Mrad 9活性的结构基础，并可能影响放疗以及理解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.