Analyzing VDJ recombination at the single molecule level

单分子水平分析 VDJ 重组

• 批准号: 6756254
• 负责人: DAVID B. ROTH
• 金额: $ 22.66万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6756254
• 关键词: DNA; T cell receptor; biophysics; biotechnology; cell differentiation; gene mutation; gene rearrangement; gene targeting; genetic techniques; immunoglobulin genes; lymphocyte; molecular genetics; nanotechnology; protein purification; protein structure function

DESCRIPTION (provided by applicant): V(D)J recombination, a site-specific DNA rearrangement reaction, is responsible for assembling immunoglobulin and T cell receptor variable region genes during lymphocyte differentiation, and it is essential for the generation of B and T lymphocytes. Aberrant V(D)J recombination events underlie a considerable fraction of lymphoid neoplasms, which are among the most common cancers. The actions of a single molecule are of utmost importance in these reactions, yet they elude the approaches available to us through "bulk biochemistry." No one has yet applied single-molecule studies to site-specific recombination; the studies we propose herein will be the first such studies conducted to understand DNA rearrangement using single-DNA micromanipulation techniques. Two specific DNA-protein complexes perform key regulatory functions in the V(D)J recombination reaction. The synaptic complex, comprising the RAG-1 and RAG-2 proteins plus accessory molecules, brings together the two DNA segments that are to undergo recombination. After DNA cleavage, the RAG proteins remain associated with the broken DNA ends in the form of a post-cleavage complex that helps direct proper joining. These two RAG-DNA complexes are critical to safeguarding the genome, yet we know little about their formation or specific activities. We propose to develop new biophysical tools to directly observe and control their formation. Specifically, we will 1) construct the necessary DNA substrates and a molecular tweezer apparatus; 2) analyze V(D)J synaptic complex formation on single tethered DNA molecules; and 3) analyze the post-cleavage complex using single-DNA studies.

描述（申请人提供）：V（D）J重组是一种位点特异性DNA重排反应，在淋巴细胞分化过程中负责组装免疫球蛋白和T细胞受体可变区基因，并且对于B和T淋巴细胞的产生至关重要。异常的V（D）J重组事件是相当一部分淋巴肿瘤的基础，淋巴肿瘤是最常见的癌症之一。在这些反应中，单个分子的作用是至关重要的，然而它们却避开了我们通过“整体生物化学”所能获得的方法。“还没有人将单分子研究应用于位点特异性重组;我们在此提出的研究将是第一个使用单DNA显微操作技术来理解DNA重排的研究。两个特定的DNA-蛋白质复合物在V（D）J重组反应中执行关键的调节功能。突触复合物，包括RAG-1和RAG-2蛋白加上辅助分子，将两个DNA片段结合在一起进行重组。在DNA切割后，RAG蛋白以切割后复合物的形式与断裂的DNA末端保持结合，这有助于指导正确的连接。这两个RAG-DNA复合物对于保护基因组至关重要，但我们对它们的形成或具体活动知之甚少。我们建议开发新的生物物理工具来直接观察和控制它们的形成。具体而言，我们将1）构建必要的DNA底物和分子镊子装置; 2）分析单个拴系DNA分子上V（D）J突触复合物的形成; 3）使用单DNA研究分析切割后复合物。