Nagawa Fumikiyo, Kodama Masami, Nishihara Tadashi, Ishiguro Kei-Ichiro, Sakano Hitoshi
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
Mol Cell Biol. 2002 Oct;22(20):7217-25. doi: 10.1128/MCB.22.20.7217-7225.2002.
In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre- and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.
在抗原受体基因的V(D)J连接过程中，两个重组信号序列（RSSs），即12-RSS和23-RSS，配对并与重组激活基因RAG1和RAG2的蛋白质产物形成复合物。我们使用磁珠纯化了V(D)J连接的切割前和切割后复合物，并通过DNA酶I足迹法对其进行分析。在切割前的突触复合物中，不仅在9聚体和间隔区观察到强烈的保护作用，而且在7聚体的编码边界附近也观察到强烈的保护作用。这与单个RSS-RAG复合物形成鲜明对比，在单个RSS-RAG复合物中，9聚体在相互作用中起主要作用。我们还通过足迹法分析了切割后的信号末端复合物。与切割前复合物不同的是，整个7聚体及其相邻的间隔区都受到了保护。本研究表明，一旦形成用于切割的突触复合物，7聚体区域中的RAG-RSS相互作用就会发生剧烈变化。
