2 Role of RAG1 and RAG2 in B-cell development | 29 that abrogate RAG2’s ability to bind H3K4me3 severely impaired V(D)J recombination in vivo61. Furthermore, the junction analysis of mice bearing RAG2 with truncated C-terminus showed the formation of genomic lesions in the proximity of cRSSs, including several oncogenes103, and similar lesions have also been observed in human pre-B cells derived from B-ALL patients104. Next to the lesions observed in the proximity of the individual cRSS, the genomic events can also take place between pairs of cRSS. This may result in a chromosome translocation as seen in several cases of human pre-T cells derived from patients suffering from T-cell acute lymphoblastic leukemia (T-ALL), bearing recombination between TCR gene segments and the Ikaros locus (Ikzf1)105, neurogenic locus notch homolog protein 1 (Notch1)106, phosphatase and tensin homolog (PTEN)107 or stem cell leukemia (SCL)/ SCL interrupting locus (SIL)108,109. Besides being a sequence-specific recombinase, RAG1/2 has also been shown to recognize specific DNA structures and cleave DNA even without the presence of RSS motifs, thus acting as a structure-specific nuclease, cleaving, for instance, heterologous loops of G-quadruplexes110,111. For example, RAG1/2 shows 3’-flap endonuclease activity that is able to remove single-strand (ss) extensions from branched DNA112. In follicular lymphoma, non-B DNA structures were also identified around the BCL2 major breakpoint region (Mbr) of the t(14;18) translocation, where the BCL2 gene is juxtaposed to the Igh locus. RAG complex was shown to bind and nick at non-B DNA structures in the proximity of BCL2 Mbr, resulting in double-strand breaks in vitro, thus demonstrating the transposase activity of RAG1/2113. Simple repeat sequences have also been shown to assume non-B DNA structures and become a RAG1/2 target. For instance, CA-repeats were shown to function as a type of cryptic RSS (cRSS) as RAG1/2 was able to cleave such structures114. In addition, the GC-rich regions are also able to adopt structures such as hairpins, cruciform or triple-stranded DNA, and the GC-rich motif 5’-GCCGCCGGGCG-3’ was identified as RAG1/2 transposition hotspot115. In our study (manuscript under consideration) we identified RAG1/2-dependent DSBs on genome-wide scale by using NBS1 ChIP-seq. Though the RAG1/2 DSBs were clearly enriched at Ig light chain regions, where they associated with RSS motifs as expected, the majority of the RAG-dependent DNA breaks were found outside of the Ig loci and showed no appreciable association with cRSSs. Interestingly, simple repeat sequences such as GA and CA repeats, but also GC-rich motifs were found to be enriched in the 500-1000bp proximity of the RAG1/2-dependent DSBs, further underscoring the propensity of the repeat regions to become RAG1/2 target. Next to aberrant RAG1/2 targeting in developing lymphocytes, the aberrant or persistent expression of RAG1/2 also represents a significant threat to genome integrity, various aspects of which are outlined in the next chapters.
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