2 Role of RAG1 and RAG2 in B-cell development | 27 antigen-binding sites82. In addition, the terminal deoxynucleotidyl transferase (TdT) further augments the diversity of the junctional sequences by catalyzing the addition of the non-templated nucleotides (N-nucleotides) to the coding ends. This process of junctional diversification can potentially expand the diversity from around 106 up to 1011 possible combinations83. Typically, the ends are filled in by the Pol X family of polymerases, namely Pol μ and Pol λ. Defects in these polymerases in mice resulted in shorter Igh and Igl D to J and V to DJ junctions. Next, the Ligase IV/XRCC4 complex ligates the processed ends. This DNA repair pathway is known as classical non-homologous end joining (c-NHEJ) where Ku70, Ku80, Artemis, XRCC4, and DNA Ligase IV are considered to be indispensable for this process, conserved throughout the evolution in different cell types84. Finally, the DNA between the two recombining segments is removed and covalently sealed at the signal ends, forming a so-called excised signal circle (ESC). It has been estimated that the production of every functional antigen receptor gene can generate up to 10 ESCs, depending on the level of non-productive recombinations. ESCs are non-replicative elements that are likely to be lost or diluted in the subsequent cell divisions85,86. Aberrant V(D)J recombination and aberrant RAG mistargeting Aberrant V(D)J recombination Next to the typical outcome of the V(D)J recombination – the formation of the coding and the signal ends, alternative outcomes have been reported. For instance, formation of so-called “hybrid joints” (HJ) when the coding end of one exon is joined to the signal end of another cleaved exon. Such events can occur in a small percentage of murine and human B cells. HJs do not contribute to the repertoire diversity and their role in oncogenic transformation remains inconclusive87. Increased formation of hybrid joints was observed in mice harboring inactivating mutation in Nijmegen breakage syndrome 1 protein (NBS1). NBS1 is part of the MRE11-RAD50-NBS1 complex (MRN), which plays a crucial role in DNA repair. The NBS1 mutation was shown to promote DNA repair through the alternative NHEJ pathway, known for its error-prone nature, often leading to small insertions or deletions88. In mice overexpressing coreRAG1 (cRAG1) and coreRAG2 (cRAG2) proteins, extremely high frequencies of HJ formation were observed as compared to cells derived from mice overexpressing full-length RAG1 and RAG2, suggesting that the non-core RAG regions suppress HJ formation under physiological conditions. In addition, leukemia that the mice expressing cRAG1 and cRAG2 developed was more aggressive and showed more genomic instability, as judged by the percentage of gH2AX-positive cells, which is a marker for DSBs89. However, the formation of HJ in endogenous wildtype/unmanipulated context seems to be rather rare. In other cases, the original pairs of coding and signal, cleaved by RAGs, are rejoined leading to a formation of “open-shut joints”. These are rather difficult to detect if there are
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