The MRAD9 Radioresistance Gene

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。以前的努力集中在裂变酵母S。pombe rad 9，一种基因， γ射线抗性、UV抗性、对DNA复制抑制剂羟基脲的抗性，并调节 相关的细胞周期检查点。我们鉴定了人（HRAD 9）和小鼠（Mrad 9）直系同源物， 发现相应的cDNA部分补充了由rad 9：：ura 4+证明的几种缺陷 酵母此外，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，其可以结合抗凋亡蛋白。 蛋白BCL-2和BCL-xL，并且当过表达时可引起细胞凋亡。我们还发现， 多功能蛋白可以结合p53并共调节p21。本提案的主要重点是， 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 利用在上一个资助期内构建的Mrad 9敲除细胞， 这些假说旨在阐明Mrad 9的功能，从而解释参与这些疾病的分子机制。 细胞对DNA损伤的反应这些假设包括：1）基因突变导致对 DNA损伤至少部分是由于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） 在存在或不存在外源DNA损伤剂的情况下，Mrad 9调节基因组稳定性，并且它是 通过重组蛋白的作用介导; 3）新发现的结构和功能 Rad 9的相关蛋白，称为Mrad 9 B（HRAD 9 B），也在介导抗 DNA损伤和维持基因组稳定，两种相关蛋白的活性部分被 多余实验方法将通过分子，细胞和 Mrad 9突变细胞的生物化学特征，以及在Mrad 9 B parkinsect中也改变的细胞。这些 研究将从分子到细胞水平检查Mrad 9的功能，确定Mrad 9的结构基础， 活性，并可能影响放射治疗以及对DNA遗传易感性的理解 损害

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 approacheswill address these hypotheses through the molecular, cellular and biochemical characterizationof 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.