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J Biol Chem. 2015 May 8;290(19):11802-17. doi: 10.1074/jbc.M115.638627. Epub 2015 Mar 5.

Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2.

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From the Departments of Immunobiology and Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, Connecticut 06511.
the Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania, and.
From the Departments of Immunobiology and Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, Connecticut 06511, the Howard Hughes Medical Institute, New Haven, Connecticut 06511


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 pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 μM) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it 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 (amino acids 997-1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 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 value for their interaction, which could help limit off-target RAG activity.


Amino Acid; Biosensor; DNA Cleavage; DNA Recombination; Protein Structure; Protein-Protein Interaction; RAG1; RAG2; V(D)J Recombination

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