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损伤可能导致突变、基因组不稳定、死亡、癌症或其他有害的健康问题。我们之前的工作集中在裂变酵母S. pombe rad9上，这是一种促进伽马射线抗性，紫外线抗性，对DNA复制抑制剂羟基脲抗性的基因，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Rad9蛋白可以结合p53并协同调节p21。最近的研究表明，Rad9还参与多种DNA修复途径。我们发现Rad9与Rad51物理相互作用并在同源重组修复中起作用，其他人报道Rad9可以结合并调节几种参与碱基切除修复和错配修复的蛋白质的活性。有趣的是，我们发现了Rad9的结构和功能相似的同源物，我们称之为HRAD9B（人类）和Mrad9B（小鼠），这表明Rad9是一个基因家族的一部分。我们构建了Mrad9和Mrad9B敲除细胞和小鼠，发现这两个基因都是胚胎发生所必需的。此外，基于Rad9的几种已知功能，如维持基因组稳定性和同源重组，这对精子发生至关重要，我们研究了Rad9是否在这一过程中起作用。我们现在提供了新的初步数据，表明我们在早期谱系a型精原细胞中构建了Mrad9靶向缺失的小鼠，并且这些动物实际上表现出精子发生缺陷。本提案的主要重点是建立并扩展我们的研究结果，以研究Rad9的功能。具体来说，我们将利用Mrad9敲除细胞和我们构建的小鼠来解决重点假设，旨在阐明该基因维持基因组稳定性、促进对DNA损伤的抵抗力和调节精子发生的机制。这些假设包括：1)Mrad9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损伤的抵抗力；2) Mrad9通过调节睾丸基因组稳定性、细胞凋亡和修复，在精子发生和减数分裂细胞周期中发挥重要作用。这些研究将从分子到细胞再到整个动物水平检测Mrad9的功能。此外，这项研究可能会对一系列重要问题产生影响，包括了解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.