Footprint Analysis of Recombination Signal Sequences in the 12/23 Synaptic Complex of V(D)J Recombination

Abstract

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.

This work was supported by grants from the Ministry of Education, Culture, and Science of Japan and the Japan Science and Technology Corporation. H.S. was also supported by Toray Science Foundation, Nissan Science Foundation, Mitsubishi Foundation, and the Foundation for Applied Enzymology. M.K. was a predoctoral fellow of the Japan Society of the Promotion of Science.

We thank Akio Tsuboi, Hirofumi Nishizumi, and Hitomi Sakano for helpful comments and Hidekazu Ishiwata, Hideaki Kikuchi, Akiko Ishikawa, and Tomoyuki Yoshida for technical assistance.

F.N. and M.K. contributed equally to this work.