V(D)J recombination, genomic instability, and cancer

描述（由申请人提供）：V（D）J重组是从单独的基因片段产生数十亿个不同的功能性抗原受体基因的过程，是淋巴细胞发育的关键步骤。然而，在其创造多样性的能力中存在错误的机会，异常重组被认为是许多白血病和淋巴瘤的原因。V（D）J重组发生在两个阶段：首先，RAG-1和RAG-2蛋白切割重组信号序列和相邻编码片段之间的DNA。由此产生的双链断裂留下平末端（信号末端）和共价密封的DNA发夹（编码末端），它们必须在过程的第二阶段连接。RAG蛋白在裂解后复合物中保留两组末端，可能有助于协调这些末端的偶联，分别形成信号接头和编码接头。连接需要非同源末端连接（NHEJ）DNA修复途径的成员。我们最近发现，加入也需要RAG蛋白本身和切割后复合物是至关重要的信号和编码联合形成。有趣的是，具有缺陷NHEJ组分的小鼠和细胞系是连接受损的，在V（D）J重组中积累断裂的DNA中间体，并且具有发展淋巴瘤的强烈倾向。在这个应用程序中，我们将描绘的RAG蛋白在加入和测试的假设，即加入缺陷RAG突变体导致淋巴细胞基因组的不稳定性，导致淋巴肿瘤的作用。本研究的具体目标如下：1）进行大规模、高通量筛选以鉴定RAG连接缺陷突变体。2)鉴定和表征连接缺陷RAG突变体的基因内抑制子。3)产生携带连接缺陷RAG突变体的转基因和敲入小鼠。4)确定RAG连接突变体对肿瘤发生的贡献。总之，这些研究将为一个强大的实验系统奠定基础，该系统将为人类常见疾病的发病机制提供许多见解，并可能允许设计诊断测试和新的治疗方法。

DESCRIPTION (provided by applicant): V(D)J recombination, the process that creates billions of distinct, functional antigen receptor genes from separate gene segments, is a crucial step in lymphocyte development. In its power to create diversity lies opportunity for error, however, and aberrant recombination has been implicated as the cause of many leukemias and lymphomas. V(D)J recombination occurs in two stages: first, the RAG-1 and RAG-2 proteins cleave DNA between the recombination signal sequences and the adjoining coding segments. The resulting double strand break leaves blunt ends (signal ends) and covalently sealed DNA hairpins (coding ends), which must be joined in the second stage of the process. The RAG proteins retain both sets of ends in a post-cleavage complex, perhaps to help orchestrate the coupling of these ends to form signal joints and coding joints, respectively. Joining requires members of the nonhomologous end joining (NHEJ) DNA repair pathway. We recently found that joining also requires the RAG proteins themselves and that the post-cleavage complex is critical for both signal and coding joint formation. Interestingly, mice and cell lines with defective NHEJ components are joining-impaired, accumulate broken DNA intermediates in V(D)J recombination, and have a strong propensity to develop lymphomas. In this application we will delineate the roles of the RAG proteins in joining and test the hypothesis that joining-deficient RAG mutants cause genomic instability in lymphocytes, leading to lymphoid neoplasms. We will pursue the following Specific Aims: 1) Perform large-scale, high-throughput screen to identify joining-deficient RAG mutants. 2) Identify and characterize intragenic suppressors of joining-deficient RAG mutants. 3) Generate transgenic and knock-in mice bearing joining-deficient RAG mutants. 4) Determine the contributions of RAG joining mutants to oncogenesis. Together, these studies will lay the foundation for a robust experimental system that should provide numerous insights into the pathogenesis of common human diseases, and may also allow design of diagnostic tests and new therapeutic approaches.