DNA-Rag protein interactions in the control of selective D gene utilization in the TCR beta locus

Ordered assembly of Ag receptor genes by VDJ recombination is a key determinant of successful lymphocyte differentiation and function. Control of gene rearrangement has been traditionally viewed as a result of complex reorganization of the nucleochromatin mediated by several nuclear factors. Selective recombination of the variable (V) genes to the diversity (D), but not joining (J), gene segments within the TCRbeta locus has been shown to be controlled by recombination signal (RS) sequences that flank the gene segments. Through ex vivo and in vitro recombination assays, we demonstrate that the Rag proteins can discriminate between the RS of the D and J genes and enforce selective D gene incorporation into the TCRbeta variable domain in the absence of other nuclear factors or chromatin structure. DNA binding studies indicate that discrimination is not simply caused by higher affinity binding of the Rag proteins to the isolated 12RS of the D as opposed to the J genes. Furthermore, we also demonstrate that the 12RS within the TCRbeta locus is functionally inferior to the consensus 12RS. We propose that selective gene segment usage is controlled at the level of differential assembly and/or stability of synaptic RS complexes, and that evolutionary "deterioration" of the RS motifs may have been important to allow the VDJ recombinase to exert autonomous control over gene segment use during gene rearrangement.