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

In mice, κ light chains dominate over λ 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 κ and λ 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 κ RSS and its flanks support six- to nine-fold higher levels of recombination than a λ counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual κ or λ RSS (plus flanks) occurs with comparable frequencies, but that a pair of κ RSS (plus flanks) support significantly higher levels of double cleavage than a pair of λ RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same κ or λ 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 κ RSS (plus natural flanks) as compared to their λ counterparts. We conclude that the overuse of κ compared to λ 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 κ 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.