196 | Appendix as CA or GA dinucleotide combinations. It is known that such sequences can adopt an alternative DNA structure, also called non-B DNA structure. These non-B DNA structures can form a triple helix or so-called G-quadruplexes, or DNA hairpin structures. Several biochemical studies have shown that the RAG1/2 complex is indeed capable of recognizing and cleaving these structures, with non-B structures identified around some genetic abnormalities commonly found in malignant B cells. In Chapters 4, 5, and 6, we describe new mechanisms of deregulation of the RAG1/2 complex. In Chapter 4, we describe a novel mechanism that causes B cells to shut down the RAG1/2 complex in response to DNA damage. This mechanism can be activated by DNA damage caused by external factors (γ-radiation, or chemical substances that introduce DNA breaks), as well as when the RAG1/2 complex itself is the source of the DNA damage. We have demonstrated that the activation of the ATM protein by DNA damage is crucial for this regulation. Pharmacological inhibition of ATM activation prevents the deactivation of the RAG1/2 complex following DNA damage induction. The DNA damage-dependent activation of ATM reduces the binding of the transcription factor FOXO1 to the RAG1 and RAG2 genes, thereby silencing the expression of these genes. We propose that this mechanism may play a significant role in protecting cells against multiple DNA damages during the gene rearrangement process. In Chapter 5, we have discovered another novel regulatory mechanism that controls the activity and availability of RAG. We show that the nuclear factor kappa-B (NF-κB) and the phosphoinositide 3-kinase (PI3K)/AKT signaling pathways are significant suppressors of RAG1/2 activity, ensuring that the RAG1/2 complex is expressed and active at the appropriate time during cell division. We demonstrate that inhibition of NF-κB results in an increase in RAG1/2 activity and RAG-mediated DNA breaks. The use of potential new therapeutic agents that inhibit NF-κB components may therefore have implications for the genomic stability of precursor B cells developing in the bone marrow. Finally, in Chapter 6, we showed that the tumor suppressor p53 also plays a crucial role in the regulation of the RAG1/2 complex in response to DNA damage. In precursor B cells lacking the p53 gene (p53 knockouts), the expression of RAG1/2 following DNA damage is not deactivated as in normal precursor B cells. Additionally, we have shown that the absence of p53 resulted in an increase in precursor B cells that have successfully undergone Ig gene rearrangement. We have demonstrated that the activation of p53 by DNA damage leads to an increase in the expression of microRNA-34a (miR-34a). miR-34a subsequently partially deactivates the transcription factor forkhead box P1 (FOXP1) protein, which plays a crucial role in the expression of the RAG1/2 complex. We have shown that DNA damage results in a reduction in FOXP1 expression, and that increased expression of FOXP1 in precursor B cells accelerates gene rearrangement.
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