V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of the recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residues 384 -1008 of 1040) alone displays binding specificity for the conserved heptamer and nonamer sequences of the RSS. The nonamer-binding region lies near the N terminus of core RAG1, whereas the heptamer-binding region has not 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 topologically independent domains within core RAG1, referred to as the central domain (residues 528 -760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span the remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant binding site for RAG2. The Cterminal 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 RAG1.The immune system displays remarkable specificity and diversity in its ability to recognize and eliminate foreign antigens. The basis for this immense diversity in many species is a complex rearrangement of the V (variable), D (diversity), and J (joining) gene segments that together encode the variable regions of T cell receptors and immunoglobulins (see Ref. 1 for review). This process, known as V(D)J recombination, requires the activity of a wide array of enzymes and is initiated by the lymphoid-specific recombination-activating proteins RAG1 and RAG2. The RAG proteins guide recombination events to conserved recombination signal sequences (RSSs) 1 that flank the genomic regions to be rearranged. Each RSS consists of a conserved heptamer and nonamer sequence separated by a 12-or 23-base pair spacer, the sequence of which is poorly conserved. Efficient recombination occurs generally between an RSS containing a 12-base pair spacer (12RSS) and one containing a 23-base pair spacer (23RSS), a requirement referred to as the 12/23 rule.The recombination process is often divided into two phases, the first phase of which consists of two distinct enzymatic steps catalyzed by the RAG proteins. The first step involves the binding of a RAG1-RAG2 complex to an RSS and the subsequent generation of a nick between the heptamer and its adjacent coding strand. The resulting 3Ј-OH group then performs a nucleophilic attack on the phosphodiester bond of the opposite strand. The primary products of this transesterification reaction are a covalently sealed hairpin, referred to as the coding end, and a blunt-ended 5Ј phosphorylated ...