The recombination difference between mouse κ and λ segments is mediated by a pair-wise regulation mechanism

In mice, kappa light chains dominate over lambda in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested kappa and lambda RSS flanked by their natural 5' and 3' flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a kappa RSS and its flanks support six- to nine-fold higher levels of recombination than a lambda counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual kappa or lambda RSS (plus flanks) occurs with comparable frequencies, but that a pair of kappa RSS (plus flanks) support significantly higher levels of double cleavage than a pair of lambda RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same kappa or lambda RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 kappa RSS (plus natural flanks) as compared to their lambda counterparts. We conclude that the overuse of kappa compared to lambda segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of kappa RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.