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

今年，我们继续研究RAG 1-RAG 2蛋白复合物的活性，特别关注这些蛋白质修饰的影响。几年前，我们的研究小组发现RAG 1的N-末端区域包含一个具有泛素连接酶（E3）活性的RING指结构域;该片段能够修饰RAG 1内附近的赖氨酸残基（K233）。当时，我们无法确定这种改变对RAG 1/2的DNA切割活性的影响，因为不可能纯化全长RAG 1（我们使用的是缺失N-末端区域的活性形式）。随着我们目前获得活性全长RAG 1的能力，我们已经回到研究泛素化对RAG 1/2的DNA切割活性的影响。扩展以前的结果，现在与泛素化形式的酶完全纯化，我们发现，裂解活性的刺激，由几倍。目前正在努力评估这种修饰在细胞内的效果。在一项单独的研究中（与NHLBI的Jay Chung博士合作），我们发现RAG 1在特定位点被AMP激活的蛋白激酶磷酸化，这种修饰也增加了RAG 1/2的活性，以及增加了细胞中的V（D）J重组。越来越明显的是，V（D）J重组是由几个代谢重要的途径。

This year we continued to study the activities of the RAG1-RAG2 protein complex, specifically focusing on the effects of modifications of these proteins. Some years ago, our group showed that the N-terminal region of RAG1 contains a RING finger domain with ubiquitin ligase (E3) activity; this fragment was able to modify a nearby lysine residue (K233) within RAG1. At that time we could not determine the effects of this alteration on the DNA cleavage activity of RAG1/2 , because it was not possible to purify full-length RAG1 (we were using an active form that was missing the N-terminal region). With our present ability to obtain active full-length RAG1, we have returned to studying the effect of ubiquitylation on the DNA cleavage activity of RAG1/2. Extending previous results, now with the ubiquitylated form of the enzyme fully purified, we find that cleavage activity is stimulated by several-fold. Efforts to assess the effect of this modification inside cells are under way. In a separate investigation (in collaboration with Dr. Jay Chung, NHLBI), we found that RAG1 is phosphorylated at a specific site by AMP-activated protein kinase, and that this modification also increases the activity of RAG1/2, as well as increasing V(D)J recombination in cells. It is becoming evident that V(D)J recombination is modulated by several metabolically significant pathways.