Paper Citing NAMD - Abstract
Zhang, Yu-Hang; Shetty, Keerthi; Surleac, Marius D.; Petrescu, Andrei J.; Schatz, David G.
Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2
JOURNAL OF BIOLOGICAL CHEMISTRY, 290:11802-11817, MAY 8 2015
Background: The RAG1-RAG2 interaction is critical for V(D)J recombination but is poorly understood. Results: The RAG1-RAG2 interaction has a binding constant of approximate to 0.4 m and requires only a small portion of RAG1. Conclusion: RAG1 and RAG2 interact with modest affinity using regions of RAG1 flanking the RAG1 catalytic region. Significance: Inefficient association of RAG1 with RAG2 could help limit damage to the genome. 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 (K-D approximate to 0.4 m) (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 -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 -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 approximate to 1,800 monomers of RAG1 and approximate to 15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the K-D value for their interaction, which could help limit off-target RAG activity.
