Analyzing VDJ recombination at the single molecule level

描述（由申请人提供）：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免疫球蛋白和T细胞受体可变区基因的组装，是B淋巴细胞和T淋巴细胞生成所必需的。异常的V(D)J重组事件是相当一部分淋巴样肿瘤的基础，这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，但它们却无法通过“散装生物化学”获得。目前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微操作技术来理解DNA重排的研究。两个特定的dna -蛋白复合物在V(D)J重组反应中起关键的调节作用。由RAG-1和RAG-2蛋白加上辅助分子组成的突触复合体将两个将要进行重组的DNA片段连接在一起。在DNA切割后，RAG蛋白仍然以切割后复合体的形式与断裂的DNA末端联系在一起，帮助指导正确的连接。这两种RAG-DNA复合物对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。具体来说，我们将1)构建必要的DNA底物和分子镊子装置；2)分析单链DNA分子上V(D)J突触复合体的形成；3)利用单dna研究分析裂解后复合体。

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.