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

今年，我们对RAG1-RAG2蛋白复合体的详细结构的研究取得了相当大的进展。我们成功地制备了这两种蛋白质的催化必需核心部分的复合体及其DNA底物的晶体，特别是在DNA切割反应完成后的复合体。已经获得了足够详细的结构，以能够识别催化活性部位、可能的DNA结合区和许多其他特征，包括重要突变的位置。这种结构大体上证实了五年前通过电子显微镜观察到的轮廓，但现在具有更高的分辨率和特异性。目前的工作旨在澄清DNA在络合物中的位置，并获得反应的其他阶段的结构。我们还继续研究RAG1的自动泛素化对RAG1N-末端区域内特定赖氨酸残基(K233)的影响。在发现RAG1/2的体外DNA切割活性被这种修饰刺激了几倍后，我们现在将这项工作扩展到评估细胞内完整的V(D)J重组反应。RAG1的自动泛素化也被发现对这种重组很重要，对于DNA切割和完成DNA产物重新连接的修复过程的后期阶段都是重要的。加上我们最近的工作表明，通过RAG1的磷酸化刺激重组，这些结果表明，V(D)J重组是由几个代谢上有意义的蛋白质修饰调控的。

This year our studies on the detailed structure of the RAG1-RAG2 protein complex have made considerable progress. We succeeded in preparing crystals of a complex of the catalytically essential core portions of both proteins, together with their DNA substrate, specifically the complex after completion of the DNA cleavage reaction. A structure sufficiently detailed to enable the identification of the catalytically active site, the probable DNA-binding regions, and many other features, including the locations of important mutations, has been obtained. The structure generally confirms the outlines that were visualized five years ago by electron microscopy, but now with much higher resolution and specificity. Current work is aimed at clarifying the position of DNA in the complex, and also at obtaining structures of other stages of the reaction. We have also continued our studies of the effects of auto-ubiquitylation of RAG1 on a particular lysine residue (K233) within the RAG1 N-terminal region. Having found that the in vitro DNA cleavage activity of RAG1/2 is stimulated several-fold by this modification, we have now extended this work to assess the complete V(D)J recombination reaction inside cells. RAG1 auto-ubiquitylation is found also to be important for this recombination, both for DNA cleavage and for a later stage of the repair process that completes the rejoining of the DNA products. Together with our recent work showing stimulation of recombination by phosphorylation of RAG1, these results make it evident that V(D)J recombination is modulated by several metabolically significant protein modifications.