Structural studies of the post-cleavage complex in V(D)J recombination

我们的研究项目旨在了解RAG1-RAG2蛋白复合物在分子细节上的功能，在过去的一年里取得了进展。从我们今年2月发表的晶体结构开始，我们已经能够解释所谓的12/23规则，该规则既适用于淋巴细胞中的V(D)J重组，也适用于RAG1-RAG2在体外的dna切割反应。这条规则指出，重组和加工需要一对不同的DNA位点，即所谓的12-RSS和23-RSS，它们的长度只相差一圈DNA。如果两个DNA序列完全相同，就不会发生反应。要理解一个对称的结构如何能产生如此不对称的结果并不容易。现在的结构显示，蛋白质中的一个柔性铰链被第一个要结合的DNA拉到一边，因此第二个DNA必须有不同的长度才能结合。详细的结构还使我们能够定位至少70个已知导致严重联合免疫缺陷（SCID）或称为Omenn综合征的相同疾病的变体形式的人类突变中的确切氨基酸。在许多情况下，这种结构解释了为什么突变会导致活性缺陷。RAG1-RAG2络合物在其反应周期前后的不同形式的结构的研究工作仍在继续。我们还完成了RAG1自泛素化对RAG1- rag2蛋白活性影响的研究。事实证明，泛素化对于刺激RAG1-RAG2的DNA切割活性，以及在细胞中重组的后期阶段，在实现DNA产物的最终修复和重新连接的步骤中，都是重要的。这项工作现已发表。

Our research program aimed at understanding the functions of the RAG1-RAG2 protein complex in molecular detail has advanced in the past year. Starting from our crystal structure published this February, we have been able to give an explanation of the so called 12/23 rule which applies both to V(D)J recombination in lymphoid cells and to the DNA-cutting reaction of RAG1-RAG2 in vitro. This rule states that recombination and processing require a pair of different DNA sites, a so-called 12-RSS and a 23-RSS, differing in length by one turn of DNA. If the two DNA sequences are identical, there is no reaction. It was not easy to understand how a symmetrical structure could produce such an unsymmetrical result. The structure now reveals that a flexible hinge in the protein is pulled to one side by the first DNA to be bound, so that the second DNA has to have a different length in order to bind. The detailed structure has also enabled us to locate the exact amino acid altered in at least 70 human mutations that are known to cause severe combined immune deficiency (SCID) or a variant form of the same disorder known as Omenn syndrome. In many cases, the structure explains why the mutation leads to defective activity. Work is continuing on obtaining structures of different forms of the RAG1-RAG2 complex, both before and after its reaction cycle. We also completed our study of the effects of auto-ubiquitination of RAG1 on the activity of the RAG1-RAG2 protein. Ubiquitination proves to be important both for stimulating the DNA cleavage activity of RAG1-RAG2, and for a later stage of recombination in cells, at the steps that achieve the final repair and rejoining of the DNA products. This work has now been published.