C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Abstract

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 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.

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

“…We find little γH2AX, 53BP1, ATM, Nbs1, or Ku70 at the Sμ in primary B cells expressing ΔE5 variants. Similar findings for Ku80, Xrcc4, and DNA-PKcs in CH12 cells were reported while this work was under review (42). Thus, CSR deficiency could be explained, in part, by the lack of recruitment of C-NHEJ core factors.…”

“…Because we see ∼50% reduction in Sμ occupancy by UNG after ΔE5, it may be that UNG is not limiting, which was suggested by the normal CSR Ung haploinsufficient mice and B cells (43). Alternatively, mismatch repair enzymes could compensate for UNG (26), although whether ΔE5 recruits more or less MSH2 to the Sμ is unclear (13,42). In any case, a partial requirement for E5 to recruit UNG does not prevent DSBs, and it does not explain why this domain is required for CSR.…”

Efficient AID targeting of switch regions is not sufficient for optimal class switch recombination

“…3 (or as in Wang et al 25 for the IgH locus). Antibodies used for pull down of DNA associated with H3K9Ac and 53BP1 or negative control antibody (Millipore) are indicated in the Supplementary Table 2 and were previously described 25,41 .…”

Enhanced Content of IgG in Burkitt’s Lymphoma Cells after Treatment with the Topoisomerase II Inhibitor, Lucanthone

“…We sought to mimic the deamination step by treating Burkitt lymphoma cells with lucanthone, an inhibitor of topoisomerase II [6], whose ability to create Abasic sites and double strand breaks in DNA of HeLa cells had been demonstrated [7,8]. As a caveat, we note that DNA deamination activity of AID might not alone represent its physiological function [9,10].…”