C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion.
Activation-induced cytidine deaminase (AID) introduces single-strand 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 compared with SHM in 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.
Departments of Immunology and Genomic Medicine and Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan.
Somayeh
S
Sabouri
Maki
M
Kobayashi
Nasim A
NA
Begum
Jianliang
J
Xu
Kouji
K
Hirota
Tasuku
T
Honjo
D000818:Animals; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059766:DNA End-Joining Repair; D005434:Flow Cytometry; D005785:Gene Conversion; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011995:Recombination, Genetic; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase
CSR synapse; DNA repair