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

描述(由申请人提供)：V(D)J重组是淋巴细胞发育的关键一步，它是从不同的基因片段中产生数十亿个不同的、具有功能的抗原受体基因的过程。然而，在它创造多样性的能力中存在着错误的机会，而异常重组被认为是许多白血病和淋巴瘤的原因。V(D)J重组分两个阶段进行：第一阶段，RAG-1和RAG-2蛋白在重组信号序列和相邻的编码片段之间切割DNA。由此产生的双链断裂留下了钝端(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蛋白在切割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连接，分别形成信号关节和编码关节。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信号和编码关节的形成都是关键的。有趣的是，带有NHEJ缺陷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，并有很强的患淋巴瘤的倾向。在这个应用中，我们将描述RAG蛋白在连接中的作用，并检验连接缺陷的RAG突变导致淋巴细胞基因组不稳定，从而导致淋巴肿瘤的假设。我们将追求以下具体目标：1)进行大规模、高通量的筛选，以确定连接缺陷RAG突变体。2)鉴定连接缺陷RAG突变体的基因内抑制子。3)获得带有连接缺陷RAG突变体的转基因和敲入小鼠。4)确定RAG连接突变体在肿瘤发生中的作用。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大量见解，还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.