Nucleotide level mapping of uracils in murine heavy chain switch regions shows correlation between uracilation and...

Introduction of uracils in specific regions within immunoglobulin genes by the activation-induced deaminase (AID) promotes mutations and double-strand breaks (DSBs). Although uracils are repaired through multiple DNA repair pathways, previous work has used mutations or DSBs as proxies for uracils and not mapped the uracils directly. We mapped uracils in the Ig heavy chain gene, Igh, in a murine cell line, CH12F3, undergoing class-switch recombination (CSR) using the uracil pull-down and sequencing technique. These cells undergo IgM-to-IgA switch upon expression of AID but do not undergo somatic hypermutation. We mapped uracils in cells defective in uracil repair and show that AID introduces high levels of uracils only in parts of switch-mu and switch-alpha regions and not in constant regions, the Igh variable region or the light chain genes. Furthermore, the peaks of uracilation match the previously determined distribution of switch junctions, which are representative of DSBs that cause isotype switching. This work confirms that AID creates uracils in both DNA strands and shows that there is a direct correlation between uracil creation and DSBs in the relevant switch regions. We evaluate proposed mechanisms of CSR in light of these findings and show that mapping uracils provides a fresh perspective on CSR.