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

今年，我们继续研究RAG1-RAG2蛋白质复合体的活性，特别是关注这些蛋白质修饰的影响。几年前，我们的研究小组发现，RAG1的N-末端包含一个具有泛素连接酶(E3)活性的环指结构域；该片段能够修饰RAG1中邻近的赖氨酸残基(K233)。当时我们不能确定这种改变对RAG1/2的DNA切割活性的影响，因为不可能纯化全长的RAG1(我们使用的是一种缺失N-末端区域的活性形式)。随着我们目前获得活性全长RAG1的能力，我们重新开始研究泛素化对RAG1/2 DNA切割活性的影响。扩展先前的结果，现在泛素化形式的酶完全纯化，我们发现切割活性被数倍刺激。目前正在努力评估细胞内这种改造的效果。在另一项研究中(与NHLBI的Jay Chung博士合作)，我们发现RAG1被AMP激活的蛋白激酶在特定的位置磷酸化，并且这种修饰也增加了RAG1/2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重组受到几条重要代谢途径的调节。

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.