STUDIES OF IMMUNOGLOBULIN GENE REARRANGEMENT

免疫球蛋白基因重排的研究

• 批准号: 6161957
• 负责人: M GELLERT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161957
• 关键词: DNA binding protein; cell free system; gene rearrangement; immunoglobulin genes; nucleic acid sequence

The V(D)J recombination process that assembles functional immunoglobulin and T cell receptor genes in lymphoid cells is essential for generating the diversity of the immune response. The reaction is now known to occur in two stages. In the first stage, specific double-strand breaks are made at the target sites by the RAG1 and RAG2 proteins acting together. The ends of the coding DNA sequence at these breaks are always joined back on themselves as DNA hairpins. The purified RAG proteins first bind to the recombination site in a "stable cleavage complex" that is highly resistant to competitor DNA. This complex requires both RAG proteins and both halves (heptamer and nonamer) of the recognition sequence. Binding and cleavage can be greatly stimulated by the ubiquitous HMG1 or HMG2 proteins, which are known to bind DNA non specifically and introduce a sharp bend. The reactions of the RAG proteins display chemical similarities to transposition. A stereochemical test showed that hairpins are made by a one step transesterification (like the DNA strand transfers of phage Mu transposase and HIV integrase), without a covalent protein-DNA intermediate, and more recent results have identified a special type of reverse reaction that extends the analogy. The later steps of V(D)J recombination that rejoin the broken ends have also been investigated. This part of the process is known to be relatively non-specific and to share many factors with DNA double-strand break repair. We have recently been able to reconstitute complete V(D)J recombination in a cell-free system. Incubation of a DNA substrate with the RAG proteins, followed by a second incubation with a mammalian cell fraction, leads to the formation of both coding joints and signal joints. The continued presence of the RAG proteins after cleavage is absolutely required for the joining of coding sequences, but inhibits the joining of recognition sequences. The RAG proteins evidently influence the ability of other factors to act on these two types of DNA ends. Coding joints from the cell-free reaction often contain self complementary tracts that arise from the asymmetric opening of hairpin ends (P nucleotides) just like those that are often found in junctions from in vivo recombination. The cell free reaction displays a strong preference for joining at sites of short DNA homologies, but the addition of human DNA ligase I leads to a more diverse set of junctions, similar to those found in vivo. It is possible that different ligases are used for homology-dependent and independent joining.

组装功能性免疫球蛋白的V（D）J重组过程 淋巴细胞中的T细胞受体基因对于产生 免疫反应的多样性。现在已知该反应发生在 分两个阶段在第一阶段，特定的双链断裂是 由RAG 1和RAG 2蛋白共同作用在靶位点产生。 在这些断裂处的编码DNA序列的末端总是连接在一起的 作为DNA发夹。纯化的RAG蛋白首先结合 在“稳定的切割复合物”中的重组位点， 对竞争者DNA的抗性。 这种复合物需要RAG蛋白 和识别序列的两个半部分（七聚体和九聚体）。 普遍存在的HMG 1可以极大地刺激结合和切割 或HMG 2蛋白，已知其非特异性结合DNA， 引入急剧弯曲。 RAG蛋白的反应显示 与转座的化学相似性立体化学测试表明， 发夹是通过一步酯交换反应（如DNA 噬菌体Mu转座酶和HIV整合酶的链转移），而不需要 共价蛋白质-DNA中间体，最近的结果有 发现了一种特殊类型的逆反应，扩展了类比。 V（D）J重组的后续步骤重新连接断裂的末端， 也被调查。这部分过程被认为是 相对非特异性，并与DNA双链共享许多因子 破损修复我们最近已经能够重建完整的V（D）J 在无细胞系统中重组。用以下物质孵育DNA底物： RAG蛋白，然后与哺乳动物细胞进行第二次孵育 分数，导致编码关节和信号的形成 接头.切割后RAG蛋白的持续存在是 绝对需要连接的编码序列，但抑制 识别序列的连接。RAG蛋白显然 影响其他因素作用于这两种DNA的能力 形接头. 来自无细胞反应的编码关节通常包含自我 发夹结构的不对称打开产生的互补束 末端（P核苷酸），就像那些经常在连接处发现的 从体内重组。无细胞反应显示出强烈的 偏好在短DNA同源性位点处连接，但是 加入人DNA连接酶I导致更多样化的连接组， 与体内发现的相似。可能不同的连接酶 用于同源依赖性和独立连接。