2 Role of RAG1 and RAG2 in B-cell development | 23 initiated by activation-induced cytidine deaminase (AID), an enzyme that is required for the execution of both mechanisms39–41. The exact molecular backgrounds of the CSR and SHM mechanisms are provided elsewhere42–44. AID has been shown to bind non-Ig loci and introduce collateral DNA damage throughout the genome of mature B cells 44–46. Thus, throughout their lifespan, B cells encounter numerous challenges to maintain genome integrity, highlighting the critical need for meticulous processes to safeguard genomic fidelity. RAG1, RAG2 and the molecular mechanisms of V(D)J recombination The evolution of RAG The human RAG1 and RAG2 genes are situated in close proximity on chromosome 11p (the mouse Rag1 and Rag2 are located on chromosome 2p), each containing one large exon, separated by only 8kb47. Such locus configuration is rather unusual and the lack of introns within the coding region is reminiscent of transposable elements. Indeed, the evolution studies of RAG1 and RAG2 structure and biochemical function indicate that RAGs originate from RAG-like (RAGL) transposable elements and evolved through so-called “molecular domestication” to their current form48. Transposons are considered fragments of “selfish DNA,” acting as molecular parasites capable of replicating within the host’s genome without offering any discernible benefit. These mobile genetic elements spread within genomes by excising or copying themselves from one location and inserting into another49,50. The first transposition event that led to the formation of the split antigen receptor gene occurred probably in early jawed vertebrate ancestors by invading the Ig-domain gene receptor exon by the RAGL_A family transposon. After this event, the RAGL_A transposon became a functional host protein, and its DNA-cleaving activity became essential for the expression of the split gene. A selective pressure caused an evolutionary adaptation of the RAGL_A in the way that it maintained its excision activity to allow the gene assembly, but at the same time suppressed the integrase activity of RAGL_A to limit its liability to genome integrity48,51,52. Such an evolutionary change, termed the “molecular transposon domestication”, might have occurred when the early jawed vertebrates acquired the arginine 848 mutation in RAG1L (R848). In most invertebrates, methionine would be found in this position (M848). Interestingly, the methionine to arginine mutation does not seem to increase the efficiency of DNA cleavage, but it strongly suppresses the transposition activity and improves the odds of maintaining the genome integrity during the gene recombination53,54. RAG1 and RAG2 proteins The human RAG1 contains 1043 amino acids (murine RAG1 contains 1040 amino acids) and is composed of 7 distinct structural domains. It contains a catalytic site that is responsible
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