The MRAD9 Radioresistance Gene

描述(申请人提供)：生物对辐射暴露的反应方式很重要，因为诱发的DNA损伤可能导致突变、基因组不稳定，以及死亡、癌症或其他有害的健康问题。我们之前的工作主要集中在裂解酵母S.pombe rad9上，这是一个促进抗伽玛射线、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(HRAD9)和小鼠(Mrad9)的同源基因，并发现相应的cDNA部分弥补了rad9：：ura4酵母所显示的几个缺陷。此外，我们还发现，HRAD9蛋白在其C-末端与检查点蛋白HHUS1和HRAD1结合，在其N-末端含有一个BH3样结构域，可与抗凋亡蛋白bcl2和bclxl结合，当过表达时可引起细胞凋亡。我们还发现，这种多功能的Rad9蛋白可以与p53结合，共同调节p21。最近的研究表明，Rad9还参与了多条DNA修复途径。我们发现RAD9与RAD51在物理上相互作用，并在同源重组修复中发挥作用，其他人报道了RAD9可以结合和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，我们发现了Rad9的结构和功能相似的类似物，我们称之为HRAD9B(人)和Mrad9B(鼠)，这表明Rad9是一个基因家族的成员。我们构建了Mrad9和Mrad9B基因敲除细胞和小鼠，发现这两个基因对胚胎发育都是必不可少的。此外，基于Rad9在维持基因组稳定性和同源重组等对精子发生至关重要的已有功能的基础上，我们研究了Rad9是否在这一过程中发挥作用。我们现在提供的新的初步数据表明，我们构建的小鼠在A型早期世系精原细胞中具有Mrad9的靶向缺失，并且这些动物实际上显示出精子发生的缺陷。这项建议的主要重点是建立和扩展我们的发现，以研究Rad9的功能。具体地说，我们将利用我们构建的Mrad9基因敲除细胞和小鼠来解决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的重点假说。这些假说包括：1)Mrad9通过调节特定的DNA修复途径和细胞周期检查点来提高基因组的稳定性和细胞对DNA损伤的抵抗力；以及2)Mrad9通过调节基因组的稳定性、细胞的凋亡和修复，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查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，包括了解对DNA损伤的固有易感性，以及对放射治疗的影响，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.