Footprint analysis of recombination signal sequences in the 12/23 synaptic complex of V(D)J recombination.
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.
Nagawa
Fumikiyo
F
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
Kodama
Masami
M
Nishihara
Tadashi
T
Ishiguro
Kei-Ichiro
K
Sakano
Hitoshi
H
D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D021901:DNA, Intergenic / Q000737:chemistry; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D021382:Protein Sorting Signals; D011995:Recombination, Genetic; D045567:VDJ Recombinases
D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D021382:Protein Sorting Signals; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases