The MRAD9 Radioresistance Gene

描述（由申请人提供）：生物体对辐射暴露的反应方式很重要，因为诱导的DNA损伤可能导致死亡、突变或癌症。之前的研究主要集中在裂变酵母S. pombe rad9上，这是一种促进抗伽马射线、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。我们鉴定了人类（HRAD9）和小鼠（Mrad9）同源基因，发现相应的cdna部分地补充了rad9::ura4+酵母所证明的几种缺陷。此外，我们发现HRAD9蛋白在其c端区结合检查点蛋白HHUS1和HRAD1，在其n端区含有bh3样结构域，可以结合抗凋亡蛋白BCL-2和BCL-xL，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结合p53并共同调节p21。本提案的主要重点是建立并扩展了我们使用酵母和哺乳动物系统积累的大量数据。具体来说，我们将利用在之前资助期间构建的Mrad9敲除细胞来解决旨在阐明Mrad9功能的明确假设，从而解释参与细胞对DNA损伤反应的分子机制。这些假设包括：1)基因突变导致对DNA损伤的敏感性至少部分是由于碱基切除修复的缺陷，而不仅仅是在细胞周期检查点；2) Mrad9在外源DNA损伤剂存在或不存在的情况下调控基因组稳定性，并通过重组蛋白的作用介导；3)新发现的与Rad9结构和功能相关的类似蛋白Mrad9B （HRAD9B）在介导DNA损伤抗性和维持基因组稳定性方面也发挥着重要作用，这两个相关蛋白的活性部分冗余。实验方法将通过Mrad9突变细胞和Mrad9B平行突变细胞的分子、细胞和生化特征来解决这些假设。这些研究将从分子到细胞水平检查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对DNA损伤的遗传易感性的理解。

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.