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

描述（由申请人提供）：V(D)J重组，从不同的基因片段中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也存在着出错的机会，而异常的重组已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蛋白在重组信号序列和相邻编码片段之间切割DNA。由此产生的双链断裂会留下钝端（信号端）和共价密封的DNA发夹（编码端），它们必须在该过程的第二阶段连接起来。RAG蛋白在裂解后复合体中保留了两组末端，可能是为了帮助协调这些末端的耦合，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。我们最近发现连接也需要RAG蛋白本身，并且切割后复合体对于信号和编码连接的形成都是至关重要的。有趣的是，具有缺陷的NHEJ成分的小鼠和细胞系连接受损，在V(D)J重组中积累断裂的DNA中间体，并且有很强的发展淋巴瘤的倾向。在本应用中，我们将描述RAG蛋白在连接中的作用，并验证连接缺陷RAG突变体导致淋巴细胞基因组不稳定，导致淋巴样肿瘤的假设。我们将追求以下具体目标：1)进行大规模，高通量筛选，以识别连接缺陷的RAG突变体。2)鉴定和表征连接缺陷RAG突变体的基因内抑制因子。3)产生带有连接缺陷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.