Recombination signal sequence variations and the mechanism of patterned T-cell receptor-beta locus rearrangement

Rearrangement of antigen receptor genes is controlled at multiple levels. One important regulation is achieved through variation in the recombination signals (RS) that flank the rearranging variable (V), diversity (D), and joining (J) gene segments. Several functional and biochemical studies have confirmed the importance of RS variations in gene rearrangement but very few molecular analyses has been reported with known, endogenous RS motifs. We have shown previously that rearrangement of the murine T-cell receptor (TCR)-B, D, and J genes follows a stereotypical pattern determined largely by the BJ genes and their flanking RS. Therefore, we studied the mechanism by which the endogenous BJ RS determine patterned gene rearrangement. We have compared the activity of three representative RS in transfection and in vitro DNA cleavage assays. Mutagenesis studies identified residues in various components of the RS and flanking coding ends that cooperate to determine the ultimate efficiency of recombination. Surprisingly, we find that changes in one component of the RS can be compensated by other elements to restore functional activity. DNA electrophoretic mobility shift assays (EMSA) show that a combinatorial effect of various higher order protein/RS complexes can, in part, control the efficiency of recombination. We propose that evolution of a patterned primary antigen receptor repertoire reflects the delicate interplay between various components of the RS and flanking coding end motifs resulting from the highly flexible interactions of the recombinase with its target DNA.