A novel cytidine deaminase affects antibody diversity.

It has not been determined whether AID normally acts as a DNA or RNA deaminase. In vitro, deamination of monomeric deoxycytidine was efficient (Muramatsu et al. 1999xMuramatsu, M, Sankaranand, V, Anant, S, Sugai, M, Kinoshita, K, Davidson, N, and Honjo, T. J. Biol. Chem. 1999; 274: 18470–18476Crossref | PubMed | Scopus (732)See all ReferencesMuramatsu et al. 1999). On the other hand, since AID has the highest homology with APOBEC1, it is likely an RNA editing deaminase. As AID expression is restricted to germinal centers and CSR and SHM both require AID, at least in humans, it is likely that AID has very specific targets for its action. This high degree of specificity resembles that of APOBEC1, which seems to have one unique ribo-cytidine target in the APO-B mRNA.The initial events in CSR, up to the production of spliced germline I-C transcripts in the μ, and γ, e, or α loci, are normal in AID-deficient mice. Thus, the requirement for AID must be in the postulated subsequent double-strand break and break repair to join Sμ with one of the downstream S regions. Clearly, CSR requires double-strand break repair by nonhomologous end joining as evidenced by the requirement for Ku70, Ku80, and the DNA-dependent protein kinase, SCID (12xRolink, A, Melchers, F, and Andersson, J. Immunity. 1996; 5: 319–330Abstract | Full Text | Full Text PDF | PubMed | Scopus (348)See all References, 2xCasellas, R, Nussenzweig, A, Wuerffel, R, Pelanda, R, Reichlin, A, Suh, H, Qin, X, Besmer, E, Kenter, A, Rajewsky, K, and Nussenzweig, M. EMBO J. 1998; 17: 2404–2411Crossref | PubMed | Scopus (259)See all References, 5xManis, J, Gu, Y, Lansford, R, Sonoda, E, Ferrini, R, Davidson, L, Rajewsky, K, and Alt, F. J. Exp. Med. 1998; 187: 2081–2089Crossref | PubMed | Scopus (230)See all References). These three proteins are also required for V(D)J recombination, but this process is normal in AID-deficient mice and humans. Thus, it is likely that a CSR-specific component of double-strand break repair, or of a process preceding the repair, is the target for AID. It is possible that AID acts as a DNA deaminase directly on the switch region DNA, but there is no precedent for such an activity. There is also no cogent model of how deamination of one or more C residues in the germline I-C RNAs would aid CSR. Thus, at this time there is no strong basis for suggesting that AID acts directly on the DNAs and RNAs involved in CSR. However, perhaps the most likely step for AID action in CSR is in the creation of the double-strand breaks in the Sμ and downstream S region DNA sequences. AID may be required for modification by cytidine deamination of the mRNAs encoding one or more of the enzymes involved in the process. The mRNAs for CSR-specific nuclease(s) are logical targets for AID. The deaminase specificity of APOBEC-1 is determined by a cofactor, ACF (Mehta et al. 2000xMehta, A, Kinter, M, Sherman, N, and Driscoll, D. Mol. Cell. Biol. 2000; 20: 1846–1854Crossref | PubMed | Scopus (175)See all ReferencesMehta et al. 2000). Likewise, AID, which contains the catalytic site for deamination, may require a cofactor that is specific for editing the mRNA of the nuclease(s) responsible for the breaks in CSR. One would postulate then, that the combination of AID and its cofactor would cause deamination of one or more C residues in the CSR nuclease mRNA(s) and that this editing would be required for the production of the active nuclease(s). In analogy to the editing of Apo-B by APOBEC-1, a single nuclease mRNA could give rise to two different nucleases by RNA editing via AID. Finally, the breaks would be repaired by the general nonhomologous end-joining machinery.AID is the first gene that has been found to be required for SHM in man. In mouse, AID may not be absolutely required, but it clearly enhances SHM. SHM can be divided into three stages, cellular activation accompanied by germinal center formation, mutation, and selection of B cells producing mutated, functional, and often high-affinity antibodies. The first stage appears to be normal in AID-deficient mice and humans. Germinal centers are formed and in fact, germinal centers are enlarged due to increased numbers of activated B cells. The selection stage may be defective in AID deficiency, although selection seems not required in mutating B cells in cell culture which persist in the absence of a selection process. In vivo, positive selection appears to be required. Mutating B cells are predestined to die by apoptosis, unless selected (Liu et al. 1991bxLiu, Y.-J, Zhang, J, Lane, P, Chan, E, and MacLennan, I. Eur. J. Immunol. 1991; 21: 2951–2962Crossref | PubMedSee all ReferencesLiu et al. 1991b). Thus, possibly, AID-deficient B cells may mutate, but die instantly. In culture the mutation process is uncoupled from selection, so this possibility could be directly investigated in AID-deficient mutating cell lines. As AID is expressed in B cells, not in accessory cells present in germinal centers (Muramatsu et al. 1999xMuramatsu, M, Sankaranand, V, Anant, S, Sugai, M, Kinoshita, K, Davidson, N, and Honjo, T. J. Biol. Chem. 1999; 274: 18470–18476Crossref | PubMed | Scopus (732)See all ReferencesMuramatsu et al. 1999), AID would have to be involved in creating a survival marker for B cells that have undergone mutation. Normally B cells with silent mutations can survive, so this marker would be some other than the Ig molecule. It is conceivable that the same or a similar marker is created in cells that have undergone CSR. However, there is no evidence that apoptosis is associated with CSR.The most likely stage at which AID operates in SHM is the mutation stage. SHM is proposed to proceed in the following order (Figure 3Figure 3) (Storb et al. 1998bxStorb, U, Klotz, E, Hackett, J, Kage, K, Bozek, G, and Martin, T.E. J. Exp. Med. 1998; 188: 689–698Crossref | PubMed | Scopus (51)See all ReferencesStorb et al. 1998b). A mutator factor loads onto an RNA polymerase initiating at an Ig transcription promoter, it is carried with the polymerase during transcript elongation, and deposited on the transcribed Ig DNA anywhere within 1–2 kb from the promoter. The mutator factor may be an endonuclease that creates a single-strand nick or double-strand cut. The nick or cut is then repaired by the cooperation between the mutator factor, exonuclease, and one or several DNA polymerases, resulting in point mutations and sometimes deletions and insertions. Before religation of the repaired strand(s), DNA mismatch repair appears to remove certain mutations. The mutations are fixed in the next S phase.It is unlikely that AID directly introduces mutations into the Ig DNA, as there is no target specificity for SHM. Any sequences inserted into an Ig gene can be mutated, despite certain preferences for short hot spots of SHM. The most likely targets for AID would be any of the DNA enzymes proposed to be involved in SHM (Figure 3Figure 3). By analogy to the proposed role of AID as required for activation of endonuclease(s) in CSR, AID may be involved in activating the proposed SHM mutator factor endonuclease gene, or more likely its mRNA, by deaminating one or more cytidines. The unedited mRNA would encode a protein with no or, in mouse, only low SHM activity.One of the HIGM2 patients (P18) showed a respectable level of SHM, with six of nine sequences containing mutations and an overall mutation frequency of 0.9%, while no CSR could be detected (Revy et al. 2000xRevy, P, Muto, T, Levy, Y, Geissman, F, Plebani, A, Sanal, O, Catalan, N, Forveille, M, Dufourcq-Lagelouse, R, Gennery, A, Tezcan, I, Ersoy, F, Kayserili, H, Ugazio, A, Brousse, N, Muramatsu, M, Notarangelo, L, Kinoshita, K, Honjo, T, Fischer, A, and Durandy, A. Cell. 2000; 102: 565–575Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesRevy et al. 2000). This finding suggests that the mechanisms of AID function in CSR and SHM may not be the same. (Alternatively, in man as apparently in mouse, AID-induced modification may not be essential for SHM, only for high levels of SHM. Other patients with the same mutation will have to be tested for this CSR/SHM dichotomy.) It would be most interesting to express the mutated AID gene of patient P18 in AID-deficient B cells or mice to determine whether AID acts differently in CSR and SHM. AID may have deaminase activity for different mRNAs or may associate with different specificity-determining cofactors in CSR and SHM. As the details of CSR and SHM are not known, several other models are possible for AID involvement in these processes. Further understanding of the AID function will no doubt play a significant role in the final unraveling of the generation of antibody diversity.*To whom correspondence should be addressed (e-mail: stor@midway.uchicago.edu).