Protein-DNA Interactions in V(D)J Recombination

V(D)J重组构建免疫球蛋白和T细胞受体基因的可变区 通过组成基因片段的组装来发育淋巴细胞。可能的数组 基因组装的组合是抗原结合受体序列多样性的主要基础 免疫系统中的分子。异常重组反应，如导致染色体的反应 易位，可导致淋巴系统恶性肿瘤。此外，V(D)J重组活性降低，作为一种 一种或另一种RAG蛋白的点突变会导致免疫缺陷疾病。至 了解这些疾病的分子基础，即催化V(D)J重组反应的因素 需要更好地刻画。最初的DNA定点切割反应是由V(D)J催化的 重组酶由RAG1和RAG2组成，重组激活基因编码的蛋白质。 RAG蛋白一起结合到一个保守的重组信号序列(RSS)，该序列与每个RAG蛋白相邻 基因片段，并催化RSS和相邻基因片段之间的双链切割 在一个两步机制中。导致基因片段组装的连接步骤需要额外的 无处不在的因素，包括在双链DNA断裂修复中起作用的蛋白质。广泛的目标 这一建议的目的是表征RAG蛋白与RSS的大分子组装。在我们的 最近的研究，我们已经确定了RAG1的结构域，每个结构域要么与RAG2，RSS， 或者编码基因片段。基于我们的结果，我们开发了一个模型，用于 V(D)J重组反应每一步的RAG1DNA结合域。为了测试我们的模型，我们将 进一步表征RAG1中的DNA结合域，并确定它们在每个催化反应中的重要性 参与复合反应。此外，我们将调查RAG2在以下方面的潜在监管角色 促进RAG1与RSS的关联。最后，每个RAG1的参与要求 在催化活性复合体的形成以及DNA切割活性中的结构域将是 测试过。这些研究的结果将为确定组装提供一个有价值的框架 和V(D)J重组酶的作用机制。

V(D)J recombination constructs the variable regions of immunoglobulin and T cell receptor genes in developing lymphocytes through assembly of component gene fragments. The array of possible combinations for gene assembly is the primary basis for sequence diversity of the antigen binding receptor molecules in the immune system. Aberrant recombination reactions, such as those resulting in chromosomal translocations, can lead to lymphoid malignancies. In addition, reduced V(D)J recombination activity, as a result of point mutations in one or the other RAG protein, can lead to immunodeficiency diseases. To understand the molecular basis for these diseases, the factors that catalyze the V(D)J recombination reaction need to be better characterized. The initial site-specific DNA cleavage reaction is catalyzed by the V(D)J recombinase consisting of RAG1 and RAG2, proteins encoded by the recombination-activating genes. Together the RAG proteins bind to a conserved recombination signal sequence (RSS), which borders each gene fragment, and catalyzes double-stranded cleavage between the RSS and the bordering gene fragment in a two-step mechanism. The joining steps, resulting in assembly of the gene fragments, require additional ubiquitous factors including proteins that function in double-stranded DNA break repair. The broad objective of this proposal is to characterize the macromolecular assembly of the RAG proteins with the RSS. In our recent studies, we have identified structural domains of RAG1 that each either interacts with RAG2, the RSS, or the coding gene segments. Based on our results, we have developed a model for participation of the RAG1 DNA-binding domains at each step of the V(D)J recombination reaction. To test our model, we will further characterize the DNA-binding domains in RAG1, and determine their importance at each catalytic step in the recombination reaction. In addition, we will investigate potential regulatory roles for RAG2 in facilitating the association of RAG1 with the RSS. Finally, the requirement for participation of each RAG1 domain in the formation of the catalytically-active complex, as well as in DNA cleavage activity, will be tested. Results from these studies will provide a valuable framework in the determination of the assembly and mechanism of the V(D)J recombinase.