Roles of the "dispensable" portions of RAG-1 and RAG-2 in V(D)J recombination

V(D)J recombination is initiated by introduction of site-specific double-st randed DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catal yze V(D)J recombination, although less efficiently than their full-length c ounterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and produ cts. We found that while truncated RAG proteins generate lower levels of re combination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates , termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispen sable" regions of the RAG proteins impairs proper processing of recombinati on intermediates. Furthermore, we found that removal of portions of the dis pensable regions of RAG-1 and RAG-2 affects the efficiency of product forma tion without altering the levels of recombination intermediates. Thus, thes e evolutionarily conserved sequences play multiple, important roles in V(D) J recombination.