Dissociation of in vitro DNA deamination activity and physiological functions of AID mutants Velizar Shivarov*, Reiko Shinkura*, and Tasuku Honjo† Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan Contributed by Tasuku Honjo, July 15, 2008 (sent for review May 30, 2008) Activation-induced cytidine deaminase (AID) is essential for the nation activity, CSR, and SHM. We chose a series of AID point DNA cleavage that initiates both somatic hypermutation (SHM) mutants (alanine-replacements) at residues located outside the and class switch recombination (CSR) of the Ig gene. Two alterna- domains comprising the C deamination catalytic center, and tive mechanisms of DNA cleavage by AID have been proposed: RNA those required for SHM-specificity, CSR-specificity, and nucleo- editing and DNA deamination. In support of the latter, AID has DNA cytoplasm shuttling, to avoid mutants with obvious causes of deamination activity in cell-free systems that is assumed to repre- physiological function loss (13–16). First, the mutant proteins sent its physiological function. To test this hypothesis, we gener- were synthesized in vitro by using a wheat germ cell-free system. ated various mouse AID mutants and compared their DNA deami- We analyzed the ssDNA deamination activity by using an in vitro nation, CSR, and SHM activities. Here, we compared DNA reaction with improved sensitivity by Alexa-680 substarate la- deamination, CSR, and SHM activities of various AID mutants and beling and infrared emission detection (Fig. S2). Among the found that most of their CSR or SHM activities were dispropor- mutants tested, one with alanine substituted for asparagine at tionate with their DNA deamination activities. Specifically, we position 51 (N51A) resulted in the complete loss of the ssDNA identified a cluster of mutants (H48A, L49A, R50A, and N51A) with deamination activity, compared with the same amount of wtAID low DNA deamination activity but relatively intact CSR activity. Of (Fig. 1A). Furthermore, although AID mutants at neighboring note is an AID mutant (N51A) that retained CSR function but lost sites (D45A and R50A) showed a dose-dependent DNA deami- DNA deamination activity. In addition, an APOBEC1 mutation at nation activity, N51A showed no enzymatic activity, even when N57, homologous to N51 of AID, also abolished DNA deamination the protein amount reached equimolar ratio to the substrate activity but retained RNA editing activity. These results indicate (Fig. 1B). Extended studies on the enzymatic activity of single that DNA deamination activity does not represent the physiolog- and composite mutants within the region between D45 and C55 ical function of AID. (Fig. 1C) identified a cluster of mutations between F46 and N51 that strongly reduced the DNA deamination activity, which class switch ͉ immunoglobulin gene ͉ APOBEC1 ͉ somatic hypermutation ͉ ranged from complete loss (N51A) to severe reduction (G47A, cytidine deaminases H48A, L49A, and R50A). All mutants with double or triple substitutions, including N51A, had little or no DNA deamination he activation of B cells by antigen stimulation induces their activity. Textensive proliferation in germinal centers, giving rise to The ability of wtAID to cause a mutator phenotype in E. coli somatic hypermutation (SHM) and class switch recombination is reported to be a marker for its dC to dU deamination activity (CSR) in the Ig locus. Such DNA alterations confer the antibody on ssDNA (5, 17). Therefore, we assayed the N51A mutant for memory required for effective vaccination. Activation-induced its mutagenic potential in the E. coli system. The median number cytidine deaminase (AID) is specifically expressed in activated of rifampicin-resistant colonies induced by N51A expression was B cells and is required for both SHM and CSR (1, 2). AID is a far less than that induced by wtAID and was comparable to that member of the cytidine (C) deaminase family. It possesses a C caused by vector alone or by a triple mutant in the catalytic deaminase domain that is indispensable for its physiological center, KSS (H56K-C87S-C90S), which resulted in the total loss functions, and recombinant AID catalyzes the deamination of C of any function (unpublished data) (Fig. 1D). Sequencing of the in vitro (1). There is, however, a long-standing dispute about mutational hot-spot region of the rpoB gene in rifampicin- whether AID deaminates C to uridine (U) on DNA (DNA resistant clones revealed that the N51A mutation profile was deamination model) (3–5) or on RNA (RNA editing model) indistinguishable from those of KSS and vector alone that may (6–8). The DNA deamination model is based on the observa- be attributable to an intrinsic mutagenic potential of E. coli as tions that AID induces a mutator phenotype in Escherichia coli reported (5) (Fig. S3). These assessments of DNA deamination and catalyzes the deamination of dC on single-stranded (ss) activity in the cell-free and E. coli systems clearly indicate that DNA in vitro (4, 5, 9, 10). However, AID’s structural homology N51A possesses no DNA deamination activity. with APOBEC1 (1), a well established RNA editing C deami- nase, suggests that it may edit mRNA to generate mRNAs Dissociation of DNA Deamination Activity and Physiological Function. encoding putative endonucleases or their guiding factors (7). We next assessed the CSR activity of three mutants (D45A, This view is supported by the requirement for de novo protein R50A, and N51A) that carried normal, 20%, and no DNA synthesis (11, 12) and the nucleo-cytoplasmic shuttling of AID deamination activity, respectively, compared with wtAID. We to achieve CSR (13). Thus, to clarify the mechanism by which expressed a similar level of AID and its mutant proteins in AID promotes CSR, we examined the requirement for AID to deaminate dC on ssDNA to exert its physiological CSR activity. For this purpose, we looked for loss-of-deamination mutants of Author contributions: R.S. and T.H. designed research; V.S. and R.S. performed research; AID that could still mediate CSR, because such mutants should V.S., R.S., and T.H. analyzed data; and T.H. wrote the paper. not exist if the DNA deamination activity is essential for AID The authors declare no conflict of interest. function [supporting information (SI) Fig. S1]. *V.S. and R.S. contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected]. Results and Discussion This article contains supporting information online at www.pnas.org/cgi/content/full/ DNA Deamination Activity of AID Mutants. We aimed to investigate 0806641105/DCSupplemental. the correlation between three activities of AID, ssDNA deami- © 2008 by The National Academy of Sciences of the USA 15866–15871 ͉ PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806641105 Downloaded by guest on September 29, 2021 A B 100 90 D45A 80 Wild- No AID Wild-type D45A R50A N51A 70 type % cleavage 0 85 86 27 0 60 b 50 40 26 R50A (% cleavage) 30 13 20 Deamination activity 10 N51A anti-His 0 0 0.2 0.4 0.6 0.8 1.0 Molar ratio of enzyme to substrate 140 C D WB: anti-AID 160 D 120 -type 140 S ector ild S 120 V W K N51A 100 100 viable cells viable 80 9 80 60 activity (%) 40 20 60 Relative deamination 0 40 A 4A 6A 1 40 52A 5 55A 51A Mutants per 10 D45AF46A H48AL49AR50AN51AK S53A C F4 G47A G -N -N51A A A A-N5 Wild-type 45 20 D45A-D45A-R50AD R50 R50 5 5 7 45A- D 0 Vector Wild-type KSS N51A Fig. 1. In vitro DNA deamination activity of AID and its mutants. (A) Representative polyacrylamide gel electrophoresis analysis of the deamination activity by AID and its mutants. The 13- and 26-base bands indicate the product (deaminated and cleaved) ssDNA and substrate ssDNA, respectively. Western blot analysis shows that comparable amounts of AID protein were used. (B) Titration of AID and its mutants. All reactions contained 5 nM oligonucleotide and proteins at the indicated enzyme/substrate molar ratios, and each assay was repeated more than three times. (C) The relative deamination activity of each mutant was determined as a percentage of the wtAID activity at the enzyme concentration showing 50% cleavage by wtAID. Bars above columns represent mean Ϯ SD. (D) Frequencies of RifR mutants generated after overnight culture of E. coli BL21 carrying an expression plasmid for AID, its mutants, or a vector control in the presence of isopropyl ␤-D-thiogalactoside. Each point represents the RifR colony number per 109 viable cells from an independent overnight culture. The median number of RifR colonies is indicated. Western blot analysis of whole lysates (107 viable cells) shows that the protein amounts of the mutant AIDs were not less than that of wtAID. AIDϪ/Ϫ spleen B cells by retroviral gene transfer. We estimated after retroviral gene transfer, we extracted the genomic DNA the percentage of IgG1ϩ-infected cells, 72 h after culturing, with and sequenced the GFP gene, as described (16) (Fig. 2D). All of LPS and IL-4 (Fig. 2A). Surprisingly, the deamination-defective the mutants showed Ͻ20% of the SHM activity of wtAID except mutant N51A showed half the WT level of CSR activity. for R50A (30%), C55A (34%), and S53A (120%). Furthermore, although D45A had normal DNA deamination DNA deamination, CSR, and SHM activities of each AID activity, it showed a very weak CSR activity. In contrast, R50A, mutant relative to wtAID are summarized in Fig. 2E. The results which had only 20% of the wtDNA deamination activity, showed indicated that most of the mutants lost the three activities even stronger CSR activity than did wtAID.