GSE39108
UNG shapes the specifity of AID-induced somatic hypermutation in non B cells
Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development. Overall design: Next Generation Sequencing analysis of mutations introduced by AID in non B cells. NIH-3T3 cells were co-transduced with mOrangeSTOP and AID-ER–expressing vectors, together with Ugi (UNG inhibitor), UNG, or empty vector as control (n=3). Transduced cells were cultured in the presence of OHT during 11 d. AID-E58Q-ER vector (catalytically inactive form of AID) was used as a negative control in combination with the previously described constructions (n=3).