Mapping and Quantitation of the Interaction Between the Recombination Activating Gene Proteins RAG1 and RAG2 [DNA and Chromosomes]

March 5th, 2015 by Zhang, Y.-H., Shetty, K., Surleac, M. D., Petrescu, A. J., Schatz, D. G.

The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pull down assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ~0.4 μM). Using the Hermes transposase as a guide, we constructed a 36 kD "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and residues N-terminal to it, but lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (aa 997-1008) near the RAG1 C-terminus and a region of RAG1 from aa 479-559. Two adjacent acidic amino acids in this region (D546 and E547) are important for both the RAG1-RAG2 interaction and recombination activity, with D546 of particular importance. Structural modeling of Mini-RAG1 suggests that D546/E547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative western blotting allowed us to estimate that mouse thymocytes contain on average ~1,800 monomers of RAG1 and ~15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD for their interaction, which could help limit off-target RAG activity.