RAG2 non-core domain regulates multiple functions of the V(D)J recombinase.

V(D)J recombination of lymphocyte antigen receptor genes is initiated by endonuclease activity of RAG1 and RAG2, termed the V(D)J recombinase. Proper regulation of the V(D)J recombinase is essential to prevent aberrant recombination events that could lead to genomic instability and lymphomagenesis. Both RAG1 and RAG2 consist of core and non-core regions, where the core regions are essential for DNA cleavage. The non-core region of RAG2 regulates multiple aspects of cellular properties of the V(D)J recombinase, although the mechanistic basis for these regulatory functions is poorly understood. We hypothesize that different regions of non-core RAG2, including the acidic hinge, plant homeodomain (PHD), and C-terminal basic region, function cooperatively to regulate V(D)J recombination activity under varying cell conditions. To test this, we examined cellular properties of RAG2 containing combinations of mutations in multiple non-core regions. Our results show a synergistic effect of the acidic region and residues in the basic region in regulating DNA cleavage activity. Further combination of mutations demonstrates the non-core region of RAG2 is highly tuned to partition the V(D)J recombinase to membrane-less DNA poor regions under normal conditions, and re-localize the recombinase in response to excess DNA damage. We propose the combination of these functions helps prevent off-target recombination and inhibits further DNA cleavage activity in the face of unrepaired DNA breaks.