The MRAD9 Radioresistance Gene

MRAD9 放射抗性基因

基本信息

批准号：
8245890
负责人：
HOWARD B. LIEBERMAN
金额：
$ 33.47万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-12-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8245890
关键词：
Address Aftercare Age Amino Acid Sequence Homology Animals Apoptosis Apoptotic Award BCL-2 Protein BCL1 Oncogene Base Excision Repairs Binding Biological Biological Assay Biological Process Bromodeoxyuridine C-terminal Cancer Etiology Cell Count Cell Cycle Cell Cycle Checkpoint Cell Cycle Progression Cell Cycle Regulation Cell Death Cell Differentiation process Cell Line Cell Proliferation Cell physiology Cells Cessation of life Chemical Exposure Chemicals Chromosome Pairing Chromosome abnormality Chromosomes Complement Complementary DNA Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA Single Strand Break DNA biosynthesis DNA lesion DNA repair protein Data Defect Detection Development Elements Embryo Embryonic Development Engineering Ethyl Methanesulfonate Event Excision Repair Exposure to Family member Fathers Female Fertility Fission Yeast Frequencies Funding Gamma Rays Gene Deletion Gene Family Gene Targeting Genes Genetic Genetic Epistasis Genetic Recombination Genome Genome Stability Genomic Instability Germ Cells Germ Lines Goals Grant Health Human Human Cloning Individual Infertility Investigation Ionizing radiation Knock-out Knockout Mice Lead Link Litter Size MLH1 gene Male Infertility Male Sterility Malignant Neoplasms Malignant neoplasm of prostate Mammals Measures Mediating Meiosis Mismatch Repair Mitomycins Molecular Morphology Mus Mutant Strains Mice Mutation N-terminal Null Lymphocytes Organism Pancreas Partner in relationship Pathway interactions Phase Phenotype Play Population Predisposition Process Protein Binding Proteins Radiation Radiation induced damage Radiation therapy Radiation-Induced Cataract Regulation Relative (related person)Reporting Research Personnel Resistance Roentgen Rays Role Sister Chromatid Exchange Site Small Interfering RNA Specimen Sperm Count Procedure Spermatocytes Spermatogenesis Spermatogonia Staging Synaptonemal Complex TdT-Mediated dUTP Nick End Labeling Assay Testing Testis Time Transgenic Mice Uracil Work Yeasts base cell motility design embryonic stem cell homologous recombination human APEX1 protein hydroxyurea in vivo inhibitor/antagonist irradiation knock-down male micronucleus mouse model mutant novel novel strategies overexpression paralogous gene prevent promoter protein complex protein protein interaction rad9 protein radiation effect recombinase recombinational repair repaired research study response sperm cell sperm morphology uptake

项目摘要

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.

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病变会导致突变，基因组不稳定性和死亡，癌症或其他有害健康问题。我们的先前努力集中在裂变酵母S. pombe rad9上，该基因促进了伽马射线耐药性，抗紫外线抗性，对DNA复制抑制剂羟基脲的耐药性，并调节相关的细胞周期检查点。我们确定了人（HRAD9）和小鼠（MRAD9）直系同源物，发现相应的cDNA部分补充了Rad9 :: ura4+酵母所证明的几种缺陷。此外，我们发现HRAD9蛋白在其C末端区域结合了检查点蛋白HHUS1和HRAD1，并在其N末端区域包含一个类似BH3的结构域，可以结合抗凋亡蛋白Bcl-2和Bcl-XL，并可能导致过度表达的凋亡。我们还发现，这种多功能RAD9蛋白可以结合p53并共同调节p21。最近的研究表明，RAD9也参与了多个DNA修复途径。我们发现RAD9与RAD51物理相互作用以及在同源重组修复中的功能，而其他人则报告说，Rad9可以结合和调节几种涉及基础切除修复和不匹配修复的蛋白质的活性。有趣的是，我们在结构和功能上相似的RAD9识别，我们称之为HRAD9B（人）和MRAD9B（小鼠），表明RAD9是基因家族的一部分。我们构建了MRAD9和MRAD9B敲除细胞和小鼠，发现这两个基因对于胚胎发生至关重要。此外，基于RAD9的几个既定功能，例如维持基因组稳定性和同源重组的作用，这对于精子发生至关重要，我们研究了RAD9是否在此过程中起作用。现在，我们提供了新的初步数据，表明我们在早期谱系精子中构建了MRAD9靶向缺失的小鼠，A型A型，实际上这些动物在精子发生中表现出缺陷。该提案的主要重点是基于我们的发现，以研究RAD9功能。具体而言，我们将利用MRAD9基因敲除细胞和我们构建的小鼠来解决旨在阐明基因维持基因组稳定性，促进对DNA损伤的机制的聚焦假设，并调节了精子发生。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来促进基因组稳定性和对DNA损伤的细胞抗性； 2）MRAD9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。这些研究将检查从分子到细胞到整个动物水平的MRAD9功能。此外，这项研究可能会影响一系列重要问题，包括了解对DNA损伤的固有易感性，对放射疗法的影响以及对精子发育和男性不育的遗传控制。