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

在过去的一年里，我们继续研究RAG1-RAG2蛋白复合物的活性，特别关注这些蛋白修饰的影响。几年前，我们的研究小组发现RAG1的n端区域含有一个具有泛素连接酶（E3）活性的环指结构域；这个片段能够修饰附近的赖氨酸残基。当时我们无法确定这种改变对RAG1/2的DNA切割活性的影响，因为不可能纯化全长RAG1（我们使用的是缺少n端区域的活性形式）。以我们目前获得活性全长RAG1的能力，我们又回到了这个问题。我们的第一个观察结果是全长RAG1在一种不同的泛素转移酶（E2） UbcH5的帮助下泛素化，而不是需要Cdc34的RING片段。反过来，Cdc34在全长蛋白上是无活性的，这再次揭示了与蛋白质片段一起工作所带来的不确定性。我们的第二个观察结果是RAG1/2的一些但不是所有的裂解反应都受到泛素化的调节。为了量化这些结果，有必要纯化完全修饰形式的RAG1，目前这项工作正在进行中。另一项单独的工作致力于鉴定细胞中与RAG1-RAG2复合物结合的其他蛋白质。在早些年，通过质谱法获得了候选蛋白质的初步列表，并通过共免疫沉淀研究得到了证实。这组蛋白包含了一些有趣的DNA修复因子，我们选择了其中的poly- adp -核糖聚合酶1 （PARP-1）进行进一步的研究。在体外，PARP-1修饰RAG1和RAG2，但有趣的是，RAG2仅在RAG1存在的情况下被修饰，这表明在该过程中具有显著的特异性。在细胞实验中，我们发现PARP-1抑制剂干扰部分但不是全部的V(D)J重组反应。目前正在进行这些观察。

In the past year, we have 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. 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 this problem. Our first observation was that full-length RAG1 is ubiquitylated with the help of a different ubiquitin transferase (E2) enzyme, UbcH5, than the RING fragment, which required Cdc34. Cdc34, in turn, is inactive on the full-length protein, revealing once again the uncertainties imposed by work with protein fragments. Our second observation was that some but not all cleavage reactions of RAG1/2 are modulated by ubiquitylation. In order to quantitate these results, it is necessary to purify the fully modified form of RAG1, and this effort is now under way. A separate effort is devoted to identifying other proteins that bind to the RAG1-RAG2 complex in cells. In an earlier year, a preliminary list of candidate proteins had been obtained by mass spectrometry and confirmed by co-immunoprecipitation studies. This group of proteins included some interesting DNA repair factors, among which we chose poly-ADP-ribose-polymerase 1 (PARP-1) for further study. In vitro, PARP-1 modifies both RAG1 and RAG2, but interestingly RAG2 is modified only in the presence of RAG1, implying some significant specificity in the process. In cellular experiments, we found that inhibitors of PARP-1 interfere with some but not all V(D)J recombination reactions. These observations are now being pursued.