Jl. Arbuckle et al., Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination, J BIOL CHEM, 276(40), 2001, pp. 37093-37101
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.