Evolution of Phosphorylation-Dependent Regulation of Activation-Induced Cytidine Deaminase  Uttiya Basu, Yabin Wang, Frederick W. Alt  Molecular Cell  Volume 32, Issue 2, Pages 285-291 (October 2008) DOI: 10.1016/j.molcel.2008.08.019 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 zAID Interacts with RPA In Vitro (A) Phylogenetic comparison of AID from Homo sapiens (human), Pan troglodytes (chimpanzee), Mus musculus (mouse), Xenopus laevis (amphibian), Gallus gallus (chicken), Danio rerio (zebrafish), Takifugu rubripes (pufferfish), and Ictalurus punctatus (catfish). Arrows indicate S38 in mammals, birds, and amphibian AID and aspartate 44 in fish AID. The conserved PKA site in tetrapods is indicated in bold and the conserved aspartic acid in telosts in red. (B) The zAIDWT, zAIDD44A, and zAIDG42S,D44A are represented with the mutated residues indicated. (C) Mutant-tagged zAID proteins were expressed in 293T cells, and nuclear extracts obtained were treated (+) or not treated (−) with PKA and immunoprecipitated. The immunoprecipitates were washed and assayed for zAID-RPA interactions by western blotting with anti-RPA antibodies. Presence of AID in the immunoprecipitation reaction was estimated by ssDNA deamination as previously described (Chaudhuri et al., 2003) and presented at the bottom of (C). (D) Flag-HA-tagged mAID and Flag-HA-tagged zAIDWT, zAIDD44A, and zAIDG42SD44A were expressed in 293 cells and partially purified by immunoprecipitation, incubated with PKA in the presence of γ-32P-ATP, and analyzed by SDS-PAGE followed by autoradiography (top panel). Presence of Flag-HA epitope-tagged mAID or zAID proteins in immunoprecipitates was determined via western blotting with anti-HA antibodies (bottom panel). Molecular Cell 2008 32, 285-291DOI: (10.1016/j.molcel.2008.08.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 zAID Deaminates Transcribed RGYW-Rich dsDNA in an RPA-Dependent Manner (A and B) mAIDWT, mAIDS38A, zAIDWT, and zAIDD44A were expressed in 293T cells and partially purified (through DEAE cellulose and glycerol gradient). The proteins were assayed for ssDNA deamination activity without PKA addition (A) or RPA-dependent dsDNA deamination activity after PKA addition (B). For (A) and (B), values represent the mean of three experiments; error bars represent SD. (C) Indicated WT and mutant AID proteins were expressed in 293T cells, partially purified as above, and standardized for ssDNA deamination activity. Subsequently, 50 μg of each partially purified AID protein, as present in the peak fraction of the glycerol gradient, was incubated with or without 50 units of PKA for 30 min at 37°C, followed by assay for RPA-dependent dsDNA deamination activity. Cytidine deamination activity for each protein was adjusted by subtracting the background value obtained with similarly processed cell extracts not transfected with AID. Deamination acivity is plotted as a % of total counts added. Background in individual assays varied from 0.3% to 1.5%. For (A), (B), and (C), values represent the mean from three experiments; error bars represent SD. Molecular Cell 2008 32, 285-291DOI: (10.1016/j.molcel.2008.08.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 CSR Activity of zAID Mutants (A and B) AID-deficient B cells stimulated with anti-CD40 and IL-4 were infected with retrovirus expressing tagged zAIDWT, zAIDD44A, or zAIDG42S, D44A. Expression of HA-tagged zAID proteins were analyzed by loading 50 μg of total B cell extracts on an SDS-polyacrylamide gel followed by western blotting using anti-HA antibodies (A), and the level of CSR to IgG1 was evaluated by flow cytometry (B) as previously described (Basu et al., 2005). (C) The percentage of GFP-positive cells that had undergone CSR to IgG1 from multiple experiments is indicated as plotted. Individual sets of experiments are represented by different symbols. In (C), the mean of all the experiments of a particular zAID protein is represented by a line. Molecular Cell 2008 32, 285-291DOI: (10.1016/j.molcel.2008.08.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 mAID “PhosphoMimetic” Mutants (A) The mAIDWT, mAIDS38A, and mAIDS38AT40D are represented with the mutated residues indicated. (B) Partially purified AID protein indicated were assayed for ssDNA deamination activities. (C) The RPA- and transcription-dependent dsDNA deamination activities of AIDWT and AIDS38AT40D proteins were analyzed in the presence (+) or absence (−) of PKA as previously described (Basu et al., 2005). (D) CSR activities of mAIDWT, mAIDS38A, and mAIDS38At40D mutants in ex vivo complementation assay. Assays are presented in the same format as corresponding assays in Figures 2 and 3 and Figure S6. In (B), (C), and (D), values represent the mean from three experiments; error bars represent SD. Molecular Cell 2008 32, 285-291DOI: (10.1016/j.molcel.2008.08.019) Copyright © 2008 Elsevier Inc. Terms and Conditions