Protein-DNA Interactions in V(D)J Recombination

V(D)J 重组中的蛋白质-DNA 相互作用

• 批准号: 6799213
• 负责人: Karla K Rodgers
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6799213
• 关键词: Protein; DNA; Interactions; Recombination

DESCRIPTION (provided by applicant): 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.

描述(由申请人提供)： V(D)J重组通过组装组成基因片段构建发育中淋巴细胞免疫球蛋白和T细胞受体基因的可变区。基因组装的一系列可能的组合是免疫系统中抗原结合受体分子序列多样性的主要基础。异常的重组反应，如那些导致染色体易位的反应，可能会导致淋巴系统恶性肿瘤。此外，由于一种或另一种RAG蛋白的点突变，V(D)J重组活性降低，可能导致免疫缺陷疾病。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。最初的DNA定点切割反应是由重组激活基因编码的RAG1和RAG2组成的V(D)J重组酶催化的。RAG蛋白一起结合到一个保守的重组信号序列(RSS)上，该序列与每个基因片段相邻，并以两步机制催化RSS和相邻基因片段之间的双链切割。连接步骤，导致基因片段的组装，需要额外的普遍存在的因素，包括在双链DNA断裂修复中起作用的蛋白质。这项建议的广泛目标是表征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，每个结构域要么与RAG2、RSS相互作用，要么与编码基因片段相互作用。基于我们的结果，我们建立了一个模型，说明RAG1DNA结合域在V(D)J重组反应的每一步中的参与。为了测试我们的模型，我们将进一步表征RAG1中的DNA结合域，并确定它们在重组反应中每个催化步骤的重要性。此外，我们将研究RAG2在促进RAG1与RSS联系方面的潜在调控作用。最后，将测试每个RAG1结构域参与催化活性复合体的形成以及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。