The recombination-activating genes, RAG1 & RAG2, play an essential role in lymphoid cell V(D)J recombination, which affects the production of highly diverse antibodies and T cell receptors (TCRs) in B and T cells. We review the background information, research insights, and applications of RAG1 & RAG2, hoping to provide helpful information for your scientific innovation.
We know that immunoglobulins (Ig) produced by B cells – integrated into both B cell surface receptors (BCR) and antibodies - and T cell surface receptors (TCR) on T cells are specific: an Ig or TCR protein can specifically recognize only one antigen. However, overall, these proteins have enormous diversity achieved by V(D)J recombination. V(D)J recombination is a somatic recombination mechanism during early T and B cell developmental maturation in vertebrates that generates diverse immunoglobulins and TCRs, which recognize different antigens by reassortment of variable (V), diversity (D), and joining (J) genes. Moreover, recombination activating genes (RAGs) RAG1 and RAG2 respectively encode the RAG-1 and RAG-2 recombinases. RAG1 and RAG2 are restrictively expressed in developing lymphocytes and are essential components of the adaptive immune system.
The recombination signal sequence (RSS) is present on both ends of the V, D, and J regions in germline genes for Ig and TCR. Structurally, the RSS has a 7 bp sequence (heptamer) at one end and a 9 bp sequence (nonamer) at the other, with a 12 bp or 23 bp spacer sequence sandwiched between them. Therefore, the RSS can be divided into two types: 12-RSS with a 12 bp spacer, and 23-RSS with a 23 bp spacer (Figure 1a). The 12/23 rule means that during V(D)J recombination, 12-RSS can only be combined with 23-RSS to ensure the correct joining of gene fragments. As an example, in germline genes of Ig heavy chain (IgH), the VH and JH genes are flanked by 23-RSS, while the DH gene is flanked by 12-RSS, so the 12/23 rule prevents direct joining of VH and JH genes, ensuring that the DH gene is involved in rearrangements (Figure 1b). Furthermore, from a molecular level, 12-RSS and 23-RSS binding also enable the formation of a more stable synaptonemal complex (synapsis).
During V(D)J recombination, the RAG1/2 complex - with the assistance of high mobility group box 1 (HMGB1), which assists DNA binding - precisely introduces a single-strand nick at the junction of the heptamer and the coding-end of the RSS. The incision generates a free 3 '- OH group at the end of the coding sequence that subsequently attacks the phosphodiester bond in the antiparallel strand to form a hairpin structure as well as a blunt signal end. The signal ended remains bound to the RAG1/2 complex and constitutes a transient structure of a "post cleavage complex." Subsequently, both the coding and signal end undergo processing and ligation by the non-homologous end joining (NHEJ) pathway to form the coding joint and the circularized signal joint (Fig.1c).
Fig. 1. V(D)J rearrangements 
(a) RSS structure: 12-RSS and 23-RSS: 7 bp heptamer and 9 bp nonamer sandwiched with a 12 bp spacer or a 23 bp spacer, respectively.
(b) The distribution of RSS in human IgH, IGK, IGL, as well as TCR α, β, γ, and δ chain genes.
(c) V(D)J recombination mechanisms.
What are the functional differences between RAG1 and RAG2? Dr. Akamatsu's experiments show that in the presence of RSS and nonspecific DNA sequences, RAG-1 binding to RSS is only 3 to 5 times higher than that of nonspecific DNA sequences. Alternatively, for RAG-2, no binding to DNA was observed experimentally. However, when RAG-1 and RAG-2 are present, they can form a RAG1/2 complex with DNA, which is more stable and specific than the RAG1-DNA complex and has cleavage activity in V(D)J recombination. These results illustrate that RAG2 may function in V(D)J rearrangements to assist RAG1 recognition of DNA and that efficient binding versus recognition of the RSS requires the concomitant presence of both RAG-1 and RAG-2 .
Both RAG-1 and RAG-2 recombinases are critical for the development and maturation of T and B cells and the production of immunoglobulin. In V(D)J recombination, the RAG1/2 complex is involved in the recognition and splicing of RSS. Thus, in contrast to mice with PrkdcSCID gene mutation, Rag1, and Rag2 knockout (KO) mice lack functional T and B cells and immunoglobulins because of the disruption in their V(D)J rearrangement pathways and do not experience immune leakage ("leakiness"). The incidence of immune leakage varies among SCID mice of different genetic backgrounds. In general, SCID mice show high leakage rates on the C57BL/6J and BALB/c backgrounds, lower on a C3H background, while being extremely low on the NOD background. However, the molecular mechanisms that generate the immune leakage remain unclear currently.
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 Nishana, M., & Raghavan, S. C. (2012). Role of recombination activating genes in the generation of antigen receptor diversity and beyond. Immunology, 137(4), 271–281.
 Akamatsu Y, Oettinger MA. (1998). Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences. Mol Cell Biol, 18(8):4670-8.
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