Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

ABSTRACT

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

ACKNOWLEDGMENTS

We thank Yoshiharu Matsuura and Masaki Kashiwada (National Institute of Infectious Diseases, Tokyo, Japan) for helpful advice, Taiji Itoh, Masami Kodama, and Satomi Shichijo for expert technical assistance, and Michiko Kimura for excellent secretarial work.

The first three authors contributed equally to this work.

This work was supported by the Special Promotion Research Grant of Monbusho in Japan and by grants from Torey Science Foundation, Nissan Science Foundation, and Mitsubishi Foundation. A.J.O. was a recipient of fellowships from Monbusho and the Japan Society for the Promotion of Science.