Activation-induced cytidine deaminase (AID) introduces singlestrand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC comparedwith SHMin chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.
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