STUDIES OF IMMUNOGLOBULIN GENE REARRANGEMENT

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 分两个阶段。在第一阶段，特定的双链断裂是 由 RAG1 和 RAG2 蛋白共同作用在靶位点产生。 这些断裂处的编码 DNA 序列的末端始终相连 回到自己身上作为DNA发夹。纯化的 RAG 蛋白首先结合 到“稳定裂解复合物”中的重组位点，该复合物高度 抵抗竞争对手的DNA。 该复合物需要两种 RAG 蛋白 以及识别序列的两半（七聚体和九聚体）。 普遍存在的 HMG1 可以极大地刺激结合和裂解 或 HMG2 蛋白，已知其非特异性结合 DNA，并且 引入急弯。 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制成的（就像 DNA 噬菌体 Mu 转座酶和 HIV 整合酶的链转移），无需 共价蛋白-DNA中间体，最近的结果已经 确定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。这部分过程已知是 相对非特异性并且与 DNA 双链共享许多因素 破损修复。我们最近能够重建完整的 V(D)J 无细胞系统中的重组。与 DNA 底物一起孵育 RAG 蛋白，然后与哺乳动物细胞进行第二次孵育 分数，导致编码接头和信号的形成 关节。裂解后 RAG 蛋白的持续存在是 连接编码序列绝对需要，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类型 DNA 的能力 结束。 无细胞反应的编码接头通常包含自身 发夹不对称开口产生的互补束 末端（P 核苷酸）就像连接处常见的末端一样 来自体内重组。无细胞反应表现出强烈的 偏好在短 DNA 同源位点连接，但是 添加人类 DNA 连接酶 I 会产生更多样化的连接点， 与体内发现的相似。可能是不同的连接酶 用于同源依赖和独立连接。

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.