Phosphorylation directly regulates the intrinsic DNA cytidine deaminase activity of activation-induced deaminase and APOBEC3G protein

The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.     

The double-edged sword of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is required for Ig class switch recombination, a process that introduces DNA double-strand breaks in B cells. We show in this study that AID associates with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) promoting cell survival, presumably by resolving DNA double-strand breaks. Wild-type cells expressing AID mutants that fail to associate with DNA-PKcs or cells deficient in DNA-PKcs or 53BP1 expressing wild-type AID accumulate gammaH2AX foci, indicative of heightened DNA damage response. Thus, AID has two independent functions. AID catalyzes cytidine deamination that originates DNA double-strand breaks needed for recombination, and it promotes DNA damage response and cell survival. Our results thus resolve the paradox of how B cells undergoing DNA cytidine deamination and recombination exhibit heightened survival and suggest a mechanism for hyperIgM type II syndrome associated with AID mutants deficient in DNA-PKcs binding.     

Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells

Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory.     

Amino-terminal phosphorylation of activation-induced cytidine deaminase suppresses c-myc/IgH translocation

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.     

Characterization of Ig gene somatic hypermutation in the absence of activation-induced cytidine deaminase

Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W = A/T, R = A/G, Y = C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL eta). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL eta-mediated mutations, 1470 H chain and 1313 kappa- and lambda-chain rearrangements from three AICDA(-/-) patients were analyzed. The Ig mutation frequency of all V(H) genes from AICDA(-/-) patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated V(H) sequences from AICDA(-/-) patients was 6.8-fold less than that of normal donors. AICDA(-/-) B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity-determining regions. A significantly reduced mutation frequency in WA motifs compared with normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase activity, suggest a role for AICDA in regulating POL eta and uracil DNA-glycosylase activity. Similar results were observed in V(L) rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes.     

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision.     

Identification of a specific domain required for dimerization of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is essential to all three genetic alterations required for generation of antigen-specific immunoglobulin: class switch recombination, somatic hypermutation, and gene conversion. Here we demonstrate that AID molecules form a homodimer autonomously in the absence of RNA, DNA, other cofactors, or post-translational modifications. Studies on serial deletion mutants revealed the minimum region between Thr27 and His56 responsible for dimerization. Analyses of point mutations within this region revealed that the residues between Gly47 and Gly54 are most important for the dimer formation. Functional analyses of these mutations indicate that all mutations impairing the dimer formation are inefficient for class switching, suggesting that dimer formation is required for class switching activity. Dimer formation and its requirement for the function of AID are features that AID shares with APOBEC-1, an RNA editing enzyme of apolipoprotein B100 mRNA.     

DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine

Activation-induced deaminase (AID) is required for both immunoglobulin class switch recombination and somatic hypermutation. AID is known to deaminate cytidines in single-stranded DNA, but the relationship of this step to the class switch or somatic hypermutation processes is not entirely clear. We have studied the activity of a recombinant form of the mouse AID protein that was purified from a baculovirus expression system. We find that the length of the single-stranded DNA target is critical to the action of AID at the Cs positioned anywhere along the length of the DNA. The DNA sequence surrounding a given C influences AID deamination efficiency. AID preferentially deaminates Cs in the WRC motif, and additionally has a small but consistent preference for purine at the position after the WRC, thereby favoring WRCr (the lowercase r corresponds to the smaller impact on activity).     

Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium

Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IkappaB kinase-dependent nuclear factor-kappaB activation pathway. H. pylori-mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori-associated gastric carcinogenesis.