170 | Chapter 7 rearrange to form the hypervariable parts of the BCR/antibody heavy chain. This is followed by the expression of the pre-B cell receptor complex consisting of the rearranged Igh and a surrogate light chain. Once the Igh locus has successfully rearranged, it becomes inaccessible, thereby preventing multiple productive locus recombinations. Subsequently, the immunoglobulin light chain (Igl) loci recombine and give rise to the hypervariable parts of the BCR/antibody light chain. In this process, immunoglobulin kappa light chain (Igk) locus recombinations precede immunoglobulin lambda light chain (Igl) locus recombinations3. RAG1/2 complex is targeted to RSS sequences in the genome, consisting of conserved heptamer and nonamer sequences separated by a less conserved spacer sequence4. The location of the RSSs is primarily adjacent to the variable (V), diversity (D), and joining (J) gene segments of (Ig) and Tcr loci, restricting RAG1/2 activity to these segments. However, sequences that resemble RSS, so-called cryptic RSS (cRSS), were identified throughout the mouse and human genomes. The presence of cRSS has been observed near the recurring genomic lesions in leukemia patients, thereby implicating the participation of RAG1/2 in the formation of the genomic lesions5–7. In Chapter 3 we investigated on a genome-wide scale, whether the RAG1/2 recombinase complex is able to introduce double-stranded DNA breaks (DSBs) in regions outside of the Ig loci. We used the Nijmegen breakage syndrome 1 (NBS1) protein as a surrogate marker for DSBs. NBS1 binds to DSBs as a part of the Mre11-Rad50-NBS1 (MRN) complex, which is integral to the recognition and repair of DSBs. NBS1 recognizes and binds to the broken DNA ends, acting as a sensor of DNA damage8. In our study, the RAG off-target activity was assessed by differential binding of NBS1 to DNA in RAG-proficient and RAG2-deficient v-Abl-transformed cell lines, lacking Artemis which is indispensable for hairpin opening formed after the DNA is nicked. In the absence of Artemis, the DSBs cannot be resolved and accumulate. This is an important feature of the experimental cell system, which we leveraged in our study to increase the resolution of the off-target RAG-induced DNA breaks that would have otherwise been resolved and repaired. The v-Abl in the used cell lines introduces a developmental block, arresting the cells in the pro-to-pre-B cell stage. At this stage, the Igl is still in the germline configuration. Treatment with Abelson-kinase inhibitors, such as STI571 (imatinib) alleviates this block, whereupon the cells begin to express RAG1, RAG2 and the gene recombination of the Igl is initiated9. This model system enables the examination of DSBs in the chromosomal context of living pre-B cells undergoing Ig recombination. The use of NBS1 chromatin immunoprecipitation (ChIP) followed by massive parallel sequencing (ChIP-Seq) to detect DSBs has been validated and employed previously to study activation-induced cytidine deaminase (AID)-dependent DNA breaks on a genome-wide scale in splenic B cells activated to undergo Ig class switch recombination10. In our study, we have identified 1489 putative RAG1/2 DSBs throughout the genome in mouse v-Abl cells, with a clear accumulation of the RAG1/2-dependent NBS1 signals at
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