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损伤可导致突变、基因组不稳定性和死亡、癌症或其他有害健康问题。我们以前的工作集中在裂变酵母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Rad 9蛋白可以结合p53并共调节p21。最近的研究表明，Rad 9也参与了多种DNA修复途径。我们发现Rad 9与Rad 51物理相互作用并在同源重组修复中发挥作用，其他人报道Rad 9可以结合并调节参与碱基切除修复和错配修复的几种蛋白质的活性。有趣的是，我们鉴定了结构和功能相似的Rad 9旁系同源物，我们称之为HRAD 9 B（人类）和Mrad 9 B（小鼠），表明Rad 9是基因家族的一部分。我们构建了Mrad 9和Mrad 9 B敲除细胞和小鼠，发现这两个基因对胚胎发育至关重要。此外，根据Rad 9的几个既定功能，如在维持基因组稳定性和同源重组中的作用，这对精子发生至关重要，我们研究了Rad 9是否在这个过程中发挥作用。我们现在提供了新的初步数据，表明我们构建的小鼠在早期谱系精原细胞，A型，Mrad 9有针对性的删除，这些动物实际上表现出精子发生的缺陷。这项建议的主要重点是建立在我们的研究结果的基础上，并将其扩展到研究Rad 9的功能。具体来说，我们将利用Mrad 9敲除细胞和小鼠，我们构建解决重点假设，旨在阐明该基因保持基因组稳定性，促进抗DNA损伤，并调节精子发生的机制。这些假设包括：1）Mrad 9通过调节特定的DNA修复途径和细胞周期检查点促进基因组稳定性和细胞对DNA损伤的抗性;和2）Mrad 9通过调节睾丸中的基因组稳定性、凋亡和修复在精子发生和减数分裂细胞周期中起重要作用。这些研究将从分子到细胞到整个动物水平检查Mrad 9功能。此外，这项研究可能会对一系列重要问题产生影响，包括理解对DNA损伤的固有易感性，对放射治疗的影响，以及精子发育和男性不育的遗传控制。

DESCRIPTION (provided by applicant): The way organisms respond to radiation exposure is important since induced DNA lesions can lead to mutation, genomic instability, and death, cancer or other deleterious health problems. Previous efforts of ours 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 Rad9 protein can bind p53 and co- regulate p21. Recent studies indicate that Rad9 also participates in multiple DNA repair pathways as well. We found that Rad9 physically interacts with Rad51 and functions in homologous recombination repair, and others reported that Rad9 can bind and regulate the activity of several proteins involved in base excision repair and mismatch repair. Interestingly, we identified structurally and functionally similar paralogues of Rad9, which we call HRAD9B (human) and Mrad9B (mouse), indicating that Rad9 is part of a gene family. We constructed Mrad9 and Mrad9B knockout cells and mice and found that both genes are essential for embryogenesis. Moreover, based on several established functions of Rad9, such as roles in maintaining genomic stability and homologous recombination, which are critical for spermatogenesis, we investigated whether Rad9 functions in this process. We now provide novel preliminary data indicating that we constructed mice bearing a targeted deletion of Mrad9 in early lineage spermatogonia, type A, and that these animals in fact demonstrate defects in spermatogenesis. The major focus of this proposal builds on and extends our findings to study Rad9 function. Specifically, we will make use of Mrad9 knockout cells and mice we constructed to address focused hypotheses designed to elucidate the mechanisms by which the gene maintains genomic stability, promotes resistance to DNA damage, and regulates spermatogenesis. These hypotheses include: 1) Mrad9 promotes genomic stability and cellular resistance to DNA damage by regulating specific DNA repair pathways and cell cycle checkpoints; and 2) Mrad9 plays an important role in spermatogenesis and in the meiotic cell cycle by regulating genomic stability, apoptosis and repair in testis. These studies will examine Mrad9 function from molecular to cellular to whole animal levels. In addition, this investigation could impact on a wide array of important issues, including understanding inherent susceptibility to DNA damage, with implications for radiotherapy, as well as the genetic control of sperm development and male infertility.