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

我们的研究计划旨在了解RAG 1-RAG 2蛋白复合物在分子细节上的功能，在过去的一年中取得了进展。从今年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，该规则适用于淋巴细胞中的V（D）J重组和RAG 1-RAG 2的体外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，它们的长度相差一圈DNA。如果两个DNA序列相同，则没有反应。很难理解一个对称的结构怎么会产生这样一个不对称的结果。现在的结构显示，蛋白质中的柔性铰链被第一个要结合的DNA拉到一边，因此第二个DNA必须具有不同的长度才能结合。详细的结构也使我们能够定位至少70种人类突变中改变的确切氨基酸，这些突变已知会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。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。目前正在继续研究获得RAG 1-RAG 2复合物在其反应周期之前和之后的不同形式的结构。我们还完成了RAG 1自身泛素化对RAG 1-RAG 2蛋白活性的影响的研究。泛素化被证明对于刺激RAG 1-RAG 2的DNA切割活性和对于细胞中重组的后期阶段都是重要的，在实现DNA产物的最终修复和重新连接的步骤中。这项工作现已出版。

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.