DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells

Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5' and 3' overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5' overhangs were removed and 3' overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells. Author summary The production of different classes of antibodies/immunoglobulins (IgM, IgG, etc.) is essential for protection against diverse pathogens and effective immunity. This cellular process is triggered by the enzyme activation-induced cytidine deaminase (AID). AID mutates DNA predominantly in antibody genes, generating different types of DNA breaks. Repair of DNA breaks initiated by AID leads to the production of different antibody classes. Erroneous repair of this damage can also lead to chromosomal translocations, a hallmark of lymphomas and other cancers. In this study, we used CRISPR/Cas9 technology to model the different types of DNA breaks physiologically produced by AID. We found that the specific structure of these DNA breaks strongly influenced how they were repaired. That is, different types of DNA breaks inform different modes of rejoining. Our findings show that not all types of DNA breaks are treated equally by genome maintenance machinery in the cell. These observations provide insight into the molecular mechanisms behind antibody-dependent immunity and lymphomagenesis.