STUDIES OF IMMUNOGLOBULIN GENE REARRANGEMENT

组装功能性免疫球蛋白的V(D)J重组过程 而淋巴细胞中的T细胞受体基因是产生 免疫反应的多样性。现在已知发生了这种反应。 分两个阶段。在第一阶段，特定的双链断裂是 由RAG1和RAG2蛋白共同作用于靶点。 编码DNA序列在这些断裂处的末端总是连接在一起的 回到他们自己的DNA发夹。纯化的RAG蛋白首先结合 结合到“稳定的裂解复合体”中的重组位点，该复合体高度 对竞争对手的DNA具有抵抗性。这种复合体需要两种RAG蛋白 和识别序列的两个半部分(七聚体和九聚体)。 无处不在的HMG1可以极大地刺激结合和切割 或HMG2蛋白，它们已知与DNA非特异性结合，并 引入一个急转弯。RAG蛋白的反应显示 化学上的相似之处在于换位。立体化学测试显示 发夹是通过一步酯交换反应(就像DNA一样)制成的 噬菌体Mu转座酶和HIV整合酶的链转移)，没有a 共价蛋白质-DNA中间体，以及更新的结果 确定了一种特殊类型的反向反应，这是类比的延伸。 V(D)J重组的后续步骤是重新连接断端 也被调查过了。这个过程的这一部分是已知的 相对非特异性，与DNA双链有许多共同的因素 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。DNA底物与 RAG蛋白，然后与哺乳动物细胞第二次孵育 分数，导致编码关节和信号的形成 关节。切割后RAG蛋白的持续存在是 对于编码序列的连接是绝对必需的，但会抑制 识别序列的连接。明显的RAG蛋白 影响其他因素作用于这两种类型DNA的能力 结束了。从无细胞反应而来的编码关节通常含有自身 发夹不对称张开形成的互补纤维束 末端(P核苷酸)就像那些经常在连接处发现的一样 来自体内重组。无细胞反应表现出很强的 偏好在短DNA同源位点连接，但 人类DNA连接酶I的加入导致了一组更多样化的连接， 与体内发现的相似。有可能不同的连接酶 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.