Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination

V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of t he recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residu es 384-1008 of 1040) alone displays binding specificity for the conserved h eptamer and nonamer sequences of the RSS. The nonamer-binding region lies n ear the N terminus of core RAGI, whereas the heptamer-binding region has no t been identified. Here, potential domains within core RAG1 were identified using limited proteolysis studies. An iterative procedure of DNA cloning, protein expression, and characterization revealed the presence of two topol ogically independent domains within core RAGI, referred to as the central d omain (residues 528-760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span t he remaining relatively uncharacterized region of core RAG1. Characterizati on of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant bi nding site for RAG2. The C-terminal domain bound DNA cooperatively but did not show specificity for either conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RA G1.