Organization of RAG1/2 and RSS DNA in the Post-Cleavage Complex

Abstract

V(D)J recombination is central to establishing a functional adaptive immune system. The large repertoire of immunoglobulins and T-cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are joined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by collaboration of the lymphoid-specific proteins RAG1 and RAG2. After cleavage, the signal ends remain tightly bound to the RAG proteins in a particularly stable Signal-End Complex (SEC).To obtain 3D structural information about RAG1/2 bound to RSS DNA, isolated and purified SEC were visualized by AFM. To better define the arrangement of the RAG proteins and RSS DNA in the complex, we used RAG1 and RAG2 fused with maltose binding protein (MBP). A wide variety of complex shapes was recorded, however, it was clear that the two DNA chains predominantly exited the SEC complex from adjacent points. The volume of the protein core was consistent with the expected mass of 500 kDa corresponding to (RAG1)2-(RAG2)2 composition. MBP protrusions could be observed on the protein particles marking the N-termini of RAG1 and RAG2. To make their appearance more noticeable, we used selective antibody labeling. Fab-labeled MBPs were clearly identified peripheral to the recombinase core. When only the RAG2 MBPs were labeled, the two DNAs most often exited together from the SEC on the opposite side to the Fabs. Consistent with this observation, when only the RAG1 MBPs were labeled, they were situated closer to the exiting DNAs.The parallel arrangement of DNA and protein subunits found by AFM is in an excellent agreement with the 3D model based on EM data.