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

在过去的一年里，我们在RAG1-RAG2蛋白结构分析方面的研究进展使我们对其功能有了更全面的了解。我们现在已经获得了代表DNA RAG1/2切割的所有阶段的详细结构，并发现了一些意想不到的特征。先前报道的重组信号序列附近几个碱基对的解离被发现是激活DNA进行切割所必需的，伴随着解离的链在DNA拉链中啮合在一起，DNA拉链中相反链的交替碱基交织在一起，并通过堆叠力稳定。这种新的结构显然比链分离的结构在非水蛋白质内部更稳定。 由于RAG1/2具有二级转座酶活性，我们还确定了它的链转移中间体的结构，在该中间体中，它捕获了目标DNA，但尚未释放产物。对于RAG，这种中间体需要比相关转座酶更多的靶DNA扭曲，这可能解释了为什么RAG是一种低效的转座酶，因为它与其作为DNA切割酶的主要活性相一致。在免疫系统细胞的背景下，转位很可能是有害的。已经提交了两篇描述这些结果的论文供出版。 由于这些结果是从截短但活性形式的RAG蛋白获得的，我们现在正在寻找更长形式的结构，它包含控制RAG活性的RAG1N末端和RAG2C末端的调节元件。

Advances in our research program on the structural analysis of the RAG1-RAG2 protein have provided a fuller understanding of its functions in the past year. We now have obtained detailed structures representing all stages of the RAG1/2 cleavage of DNA, and find some unexpected features. The previously reported unwinding of several base pairs of DNA adjacent to the recombination signal sequence, found to be necessary to activate the DNA for cleavage, is accompanied by the unwound strands meshing together in a DNA zipper with alternating bases from opposite strand interleaved and stabilized by stacking forces. This novel structure is clearly more stable in the non-aqueous protein interior than a strand-separated one. As RAG1/2 has a secondary transposase activity, we have also determined the structure of its strand-transfer intermediate in which it has captured a target DNA but not yet released the product. With RAG, this intermediate requires much more distortion of target DNA than in related transposases, probably explaining why RAG is an inefficient transposase, as is compatible with its primary activity as a DNA-cutting enzyme. Transposition in the context of immune system cells would most likely be harmful. Two papers describing these results have been submitted for publication. As these results were obtained with truncated but active forms of the RAG proteins, we are now pursuing structures of longer forms, which contain regulatory elements of the RAG1 N-terminus and the RAG2 C-terminus that control RAG activity.