Cuomo C A, Oettinger M A
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Nucleic Acids Res. 1994 May 25;22(10):1810-4. doi: 10.1093/nar/22.10.1810.
The recombinase activating genes RAG-1 and RAG-2 operate together to activate V(D)J recombination, and thus play an essential role in the generation of immune system diversity. As a first step in understanding the function of the RAG-2 protein, we have tested a series of deletion and insertion mutations for their ability to induce V(D)J joining of a variety of model substrates. Mutants were assayed for their ability to induce deletional and inversional V(D)J joining, thereby testing their proficiency at forming both signal and coding joints, and, in some cases, for their ability to carry out recombination of both extrachromosomal and integrated recombination substrates. All these reactions were affected similarly by any one mutation. Although the RAG-2 protein shows extensive evolutionary conservation across its length, we found that the carboxy-terminal portion of RAG-2, including an acidic region, is dispensable for all forms of recombination tested. In contrast, all mutations we created in the N-terminal region severely decreased recombination. Thus, the core active region required for V(D)J recombination is confined to the first three-quarters of the RAG-2 protein.
重组激活基因RAG-1和RAG-2共同作用以激活V(D)J重组，因此在免疫系统多样性的产生中发挥着至关重要的作用。作为了解RAG-2蛋白功能的第一步，我们测试了一系列缺失和插入突变体诱导各种模型底物进行V(D)J连接的能力。对突变体诱导缺失型和倒位型V(D)J连接的能力进行了检测，从而测试它们形成信号接头和编码接头的能力，在某些情况下，还检测了它们对染色体外和整合型重组底物进行重组的能力。任何一种突变对所有这些反应的影响都是相似的。尽管RAG-2蛋白在其全长范围内显示出广泛的进化保守性，但我们发现RAG-2的羧基末端部分，包括一个酸性区域，对于所测试的所有形式的重组都是可有可无的。相比之下，我们在N末端区域产生的所有突变都严重降低了重组能力。因此，V(D)J重组所需的核心活性区域局限于RAG-2蛋白的前三分之四。
