Nuclear export-dependent functions of RAG2 in the DNA damage response system

DNA损伤反应系统中RAG2的核输出依赖性功能

• 批准号: 9387569
• 负责人: Karla K Rodgers
• 金额: $ 21.72万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387569
• 关键词: Adaptive Immune System; Affect; Aneuploidy; Antigen Receptors; Antigens; Apoptosis; B-Lymphocytes; CDK2 gene; Cell Cycle; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Nucleus; Cell Survival; Cell division; Cells; Centrioles; Centrosome; Chromosome abnormality; Cytosol; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; Data; Development; Ephrin-A5; Equilibrium; Event; Fluorescence Microscopy; G1 Arrest; G1 Phase; Generations; Genes; Genetic Recombination; Genome Stability; Genomic Instability; Hybrids; Image; Immunoglobulins; Incidence; Ionizing radiation; Joints; Lead; Lymphocyte; Lymphoma; Maintenance; Measures; Mediating; Metabolic; Methods; Molecular; Molecular Biology; Mutagens; Neoplasms; Nuclear Export; Pathway interactions; Pharmaceutical Preparations; Phase Transition; Phosphorylation; Physiological; Predisposition; Process; Property; Proteins; Proteomics; Publishing; Receptor Gene; Regulation; Resolution; Role; S Phase; Site; System; T-Cell Receptor Genes; T-Lymphocyte; TP53 gene; Testing; V(D)J Recombination; Work; genome integrity; high risk; leukemia; premature; prevent; repaired; response; uncontrolled cell growth

The assembly of immunoglobulin and T cell receptor genes through VDJ recombination is central to lymphocyte development. RAG1 and RAG2 generate DNA double strand breaks (DSBs) in an intermediate step of VDJ recombination. This is a risky step that can lead to genomic instability if not tightly regulated. As the physiologic generation of DNA DSBs occurs throughout lymphocyte development, the proper resolution of these breaks in the presence of ongoing RAG endonucleolytic activity must occur before progression to the next stage of development. Moreover, DNA DSBs unrelated to VDJ recombination that are coincidently present with RAG-mediated breaks must also be appropriately resolved. It is not yet clear how the DDR and the RAG proteins coordinate their respective functions in assembly of the antigen receptor loci while maintaining genomic integrity. In our recent studies, we have shown that DNA DSBs triggers ATM-dependent nuclear export and centrosome targeting of RAG2. Relocalization of RAG2 was transient, as the pre-DNA damage localization of RAG2 was re-established following DNA repair. The central hypothesis of this project is that nuclear export and centrosome targeting of RAG2 reinforces G1-S cell cycle arrest until the damaged DNA has been repaired, resulting in increased cell survival and genomic stability. We propose this occurs through regulation of centrosome duplication, which is tightly coordinated with the onset of DNA replication in the S phase. Using a proteomic approach, we have identified specific centrosomal proteins that interact with RAG2 following DNA damage. Now, we will resolve the properties of these interactions using a combination of state- of-the-art imaging with molecular biology approaches that will identify regions of RAG2 that mediate these interactions, and their site of localization within the centrosome. In addition, to test our hypothesis regarding the role of centrosome-targeting of RAG2 following DNA damage, we will measure cell survival and V(D)J recombination fidelity in cells expressing RAG2 that is proficient versus defective in centrosome targeting. This project will elucidate mechanisms that balance lymphocyte survival with maintenance of genomic integrity during development of the adaptive immune system.

通过VDJ重组的免疫球蛋白和T细胞受体基因的组装是 淋巴细胞发育。RAG1和RAG2在中间体中产生DNA双链断裂(DSB) VDJ重组的步骤。这是一个危险的步骤，如果不严格监管，可能会导致基因组不稳定。AS DNADSB的生理生成发生在淋巴细胞发育的整个过程中，适当地分解 在存在持续的RAG内切核活性的情况下，这些中断必须发生在进展到 下一阶段的发展。此外，与VDJ重组无关的DNA DSB也巧合地存在 也必须适当地解决由碎布引起的中断。目前还不清楚解甲返乡和破布是如何 蛋白质在抗原受体基因座的组装中协调各自的功能，同时维持 基因组的完整性。在我们最近的研究中，我们已经表明DNADSB触发了ATM依赖的核 RAG2的输出和中心体靶向。RAG2的重新定位是暂时的，因为DNA前损伤 DNA修复后，RAG2的定位重新建立。这个项目的中心假设是 核输出和中心体靶向RAG2加强了G1-S细胞周期的停滞，直到损伤的DNA 被修复，从而提高了细胞存活率和基因组稳定性。我们建议这通过以下方式实现 中心体复制的调节，这与S病的DNA复制的开始密切相关 相位。使用蛋白质组学方法，我们已经确定了与RAG2相互作用的特定中心体蛋白 在DNA损伤之后。现在，我们将使用状态组合来解析这些相互作用的属性- 使用分子生物学方法进行最先进的成像，将识别RAG2中介导这些 相互作用，以及它们在中心体中的定位位置。此外，为了测试我们的假设， 中心体靶向RAG2的作用DNA损伤后，我们将测量细胞存活率和V(D)J 在表达RAG2的细胞中重组的保真度在中心体靶向方面是熟练的还是有缺陷的。这 该项目将阐明平衡淋巴细胞存活和维持基因组完整性的机制 在适应性免疫系统的发展过程中。