A English summary | 195 mation of cancer? For example, an oncogene that cannot be “turned off” due to incorrectly repaired DNA breaks, causing the cells to continuously divide. Acute lymphoblastic leukemia (ALL) is a type of blood cancer characterized by rapid expansion of malignant B and T cell precursors. Genomic instability, including translocations such as ETV6-RUNX1, BCR-ABL1, and E2A-PBX1, is a feature of ALL. Traces of RAG1/2 activity, or the presence of sequences resembling RSS, have been found in the vicinity of genomic lesions in both mouse and human B-cell malignancies, including ALL. This suggests that aberrant targeting and/or activity of RAG1/2 may play a role in the development of lymphoid malignancies. However, the precise mechanism of the malignant derailment of precursor B cells in the onset of ALL, and the exact role of the RAG1/2 complex in this process, are currently unknown. The research in this thesis focuses on detecting and characterizing the “off-target” activity of RAG1/2 and the potential consequences thereof on the genomic integrity of precursor B cells. We also meticulously studied the regulation of the RAG1/2 complex in the context of DNA damage and discovered several novel regulatory mechanisms. In Chapter 2, we extensively described the process of B-cell development and V(D)J gene rearrangement. Here, we paid particular attention to the role of DNA damage and DNA damage signaling. We discuss the molecular mechanisms involved and how the balance between DNA damage and DNA repair is maintained, as well as the possible consequences of disrupting this balance. We summarized evidence from other studies showing that a significant portion of the genetic errors in patients with B-cell acute lymphoblastic leukemia (B-ALL) exhibit characteristics of dysregulated RAG1/2 activity, which may arise due to failed RAG1/2 regulation. First, we investigated whether the activity of the RAG1/2 complex in precursor B cells is limited to Ig genes, or if it can also introduce DNA breaks elsewhere in the genome. Previous studies conducted by other research groups demonstrated that the RAG1/2 complex can bind to DNA at locations other than Ig genes. However, those studies could not conclude whether the RAG1/2 binding to the DNA also results in the formation of RAG1/2-dependent DNA breaks. In Chapter 3, we demonstrated that RAG1/2 does not only introduce DSBs at Ig genes but also at thousands of other locations in the genome. In a mouse precursor B-cell line, we identified approximately 1500 sites where RAG1/2 introduces a DNA break. In addition to the known “hot spots” on the Ig gene segments on chromosome 6 (immunoglobulin light chain genes in mice) and chromosome 12 (immunoglobulin heavy chain genes in mice), RAG1/2-dependent DNA breaks were found throughout the entire genome. RAG1/2-dependent DNA breaks were identified near genes previously associated with the development of B-cell malignancies such as Ikzf4, Abl2, Klf4, Tcf4, Tcf7, Rassf8, Mef2c, or near genes involved in DNA break repair such as Rad51ap1. We further investigated the DNA sequences in the vicinity of RAG1/2-dependent DNA breaks, showing that these breaks are predominantly found near simple repetitive sequences such
RkJQdWJsaXNoZXIy MTk4NDMw