The MRAD9 Radioresistance Gene

生物对辐射暴露的反应方式很重要，因为诱导的DNA损伤可能会导致死亡， 突变或癌症。此前的努力主要集中在裂解酵母S.pombe rad9，这是一种促进 耐伽马射线，耐紫外线，耐DNA复制抑制剂羟基脲，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)的同源基因，以及 相应的cDNA被发现部分地补充了由rad9：：ura4+证明的几个缺陷 酵母。此外，我们还发现HRAD9蛋白与检查点蛋白HHUS1和HRAD1在其 C-末端区域，在其N-末端区域含有BH3样结构域，可以结合抗细胞凋亡 BCL-2和BCL-XL蛋白，过表达时可引起细胞凋亡。我们还发现这一点 多功能蛋白可与p53结合，共同调节p21。这项提案的主要重点是建立在 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Mrad9基因敲除细胞来处理定义明确的 旨在阐明Mrad9功能的假说，从而解释参与Mrad9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； 在存在或不存在外源DNA损伤剂的情况下，Mrad9调节基因组的稳定性，并且它是 通过重组蛋白的作用；3)结构和功能上的新发现 RAD9的相关类似物，称为Mrad9B(HRAD9B)，也在介导对赤霉病的抗性中发挥重要作用 DNA损伤和维持基因组稳定性，以及两个相关蛋白的活性部分 多余的。实验方法将通过分子、细胞和 Mrad9突变细胞的生化特性，Mrad9B的细胞也发生了改变。这些 研究将从分子到细胞水平检查Mrad9的功能，确定Mrad9的结构基础 活动性，并可能影响放射治疗以及了解对DNA的遗传易感性 损坏。

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.