Protein-DNA Interactions in V(D)J Recombination

除了本文所提供的篇幅外，V（D）J重组通过组装组分基因片段在发育中的淋巴细胞中构建免疫球蛋白和T细胞受体基因的可变区。用于基因组装的可能组合的阵列是免疫系统中抗原结合受体分子的序列多样性的主要基础。异常重组反应，如导致染色体易位的那些，可导致淋巴恶性肿瘤。此外，由于一种或另一种RAG蛋白中的点突变，降低的V（D）J重组活性可导致免疫缺陷疾病。为了了解这些疾病的分子基础，需要更好地表征催化V（D）J重组反应的因素。初始位点特异性DNA切割反应由V（D）J重组酶催化，所述重组酶由重组激活基因编码的蛋白质RAG1和RAG2组成。RAG蛋白一起结合到保守的重组信号序列（RSS），其与每个基因片段邻接，并以两步机制催化RSS和邻接基因片段之间的双链切割。导致基因片段组装的连接步骤需要额外的普遍存在的因子，包括在双链DNA断裂修复中起作用的蛋白质。该提案的主要目的是表征RAG蛋白与RSS的大分子组装。在我们最近的研究中，我们已经确定了RAG1的结构域，每个结构域都与RAG2，RSS或编码基因片段相互作用。基于我们的研究结果，我们已经开发了一个模型，参与RAG1 DNA结合域在每一步的V（D）J重组反应。为了测试我们的模型，我们将进一步表征RAG1中的DNA结合结构域，并确定它们在重组反应中每个催化步骤的重要性。此外，我们还将研究RAG2在促进RAG1与RSS相关性方面的潜在调节作用。最后，将测试每个RAG1结构域参与催化活性复合物形成以及DNA切割活性的要求。这些研究结果将为确定V（D）J重组酶的组装和机制提供有价值的框架。性能现场=

EXCEED THE SPACE PROVIDED, 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. PERFORMANCE SITE ========================================Section End===========================================