3 RAG1/2 introduces double-stranded breaks at non-Ig loci | 55 Introduction During lymphocyte development in bone marrow, each lineage-committed B-cell undergoes V(D)J recombination to assemble functional gene segments that encode the heavy and the light chain variable regions of its antibody. These regions are critical for antigen recognition and antigen binding. V(D)J recombination is initiated by RAG1/2 endonuclease by binding the recombination signal sequences (RSS) flanking each variable (V), diversity (D), and joining (J) gene segment and consisting of conserved heptamer and nonamer sequences, separated by a less conserved spacer region. The cleavage of DNA by RAG1/2 endonuclease occurs in a coordinated manner, RAG1/2 first introduces a single-stranded nick between the heptamer and the gene segment. This creates a 3' hydroxyl group which then attacks the other strand and generates a double-stranded DNA break (DSB). This step is also referred to as hairpin formation because it generates a covalently sealed hairpin at the end of the gene segment1. Hairpin formation only occurs in the paired complex and is thought to take place simultaneously at the two RSSs. The two blunt RSS ends are fused by classical non-homologous end-joining (c-NHEJ) pathway in which Artemis plays a pivotal role in successful gene recombination by opening the end-associated hairpins2,3. The created DSBs are detected by the MRN complex, consisting of meiotic recombination 11 (MRE11), RAD50, and Nijmegen breakage syndrome 1 (NBS1; also known as Nibrin), which binds the DNA in the proximity of the DSBs4. B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common cancers affecting all age groups. In individuals under 20 years of age, it is the leading cause of cancer-related death. It is characterized by a high degree of genomic instability. A number of genomic aberrations have also been associated with B-ALL, including fusions such as ETV6-RUNX1, BCR-ABL1, mutations in IKZF1, ZEB2, MEF2D rearrangement or PAX5 tandem duplication5–7. In the last decades, thanks to new innovative treatment options, the survival of young B-ALL patients improved dramatically8,9. Nevertheless, the true root cause of the molecular formation of these genomic alterations remains not entirely understood. Mistargeted activity of RAG1/2 recombinase can lead to an inappropriate DNA cleavage, resulting in to DNA breaks at unintended sites. Such genomic instability events may create a favorable condition for the acquisition of an oncogenic event. Several translocations or fusions have been described in B-ALL patients that bear signs of RAG1/2 activity10, including ETV6/RUNX1 gene fusion11 or PAR1 deletion, where a conserved RSS was found near the breakpoint or E2A-PBX1 translocation that harbors non-template nucleotides reminiscent of V(D)J joint12. Previous studies showed that RAG1 and RAG2 binding to DNA in B and T cells is not exclusively limited to Ig and Tcr genes. In fact, thousands of RAG1 and RAG2 binding sites were identified throughout the genome in mouse lymphocytes. Though outside of the Ig loci the presence of RSS was shown to be a poor predictor of RAG1 binding, a robust
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