Structural studies of sequential DNA cleavage by RAG1/RAG2 proteins in V(D)J recombination

在过去的一年中，我们的研究计划取得了进一步的进展，旨在从分子细节上了解RAG 1-RAG 2蛋白复合物的功能。2015年，我们报道了RAG 1/RAG 2复合物的第一个晶体结构。这是一个重大的进步，使得第一次有可能解释V（D）J重组中所谓的12/23规则，该规则迫使它在两个不同的DNA位点上工作。但这只是蛋白质复合物的结构，没有DNA，这留下了许多关于机制的问题没有答案。有必要获得关于含有DNA的结构的详细信息，并在裂解反应的所有阶段对其进行研究。由于DNA切割在几个确定的步骤中发生，因此可以获得至少四种不同种类的RAG-DNA复合物。获得了大多数这些物种的晶体，并对其结构进行了优化。此外，我们使用冷冻电子显微镜技术来获得进一步的信息。结合这些技术，我们发现RAG介导的DNA切割的一个重要步骤是解旋邻近重组信号序列的DNA的几个碱基对。这个意想不到的步骤也可以在转座酶类的相关酶中发现。这项工作已经出版。我们还在继续我们对RAG蛋白的更长版本的工作，其中包含控制RAG活性的调控元件。

Further progress has been made in the past year in our research program aimed at understanding the functions of the RAG1-RAG2 protein complex in molecular detail. In 2015, we reported the first crystal structure of the RAG1/RAG2 complex. This was a major advance, making it possible for the first time to give an explanation of the so called 12/23 rule in V(D)J recombination, which forces it to work on two different DNA sites. But this was a structure of the protein complex alone, without DNA, which left many questions about mechanism unanswered. It was necessary to obtain detailed information on structures containing DNA, and to study them at all stages of the cleavage reaction. As the DNA cleavage occurs in several defined steps, at least four distinct species of the RAG-DNA complex can be obtained. Crystals of most of these species were obtained and their structures were refined. In addition, we used the technique of cryo-electron microscopy to obtain further information. Combining these techniques, we found that an essential step in RAG-mediated DNA cleavage is the unwinding of several base pairs of DNA adjacent to the recombination signal sequence. This unexpected step may also be found in related enzymes of the transposase class. This work has been published. We are also continuing our work on longer versions of the RAG proteins, which contain regulatory elements controlling RAG activity.