Dissociation of in vitro DNA 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 (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 . 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 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 at nation activity, N51A showed no enzymatic activity, even when N57, homologous to N51 of AID, also abolished DNA deamination the 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 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 . 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 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 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. The relative CSR disproportionately except for S53A (all intact) and double/triple activities of the WT and mutant AID proteins were semiquan- mutants including N51A (all lost). This dissociation of the three titatively confirmed by RT-PCR for ␥1 switch transcripts (Fig. activities is more clearly presented by scatter plots of the 2B). CSR activities of most (7 of 11) of the mutants were not mutants’ deamination activities with their CSR and SHM activ- proportional to DNA deamination activities, although the F46A, ities (Fig. S5 and Fig. S6, respectively). It is striking that some K52A, S53A, and C55A mutants showed a somewhat weak AID mutants showed a strong DNA deamination activity in vitro, correlation between the two activities (Figs. 1C and 2C). Ex- but their SHM activities were negligible. Of note are the single tremely surprising was the finding that a cluster of mutants, and double mutants of D45A (D45A-F46A and D45A-R50A), H48A, L49A, R50A, and N51A showed much higher relative which had extremely low SHM activity despite having strong CSR activity than expected from their DNA deamination activ- DNA deamination activity. However, whether or not the DNA ity. Mutations at residues between K52 and C55 revealed subtle deamination is involved in SHM is equivocal because the loss of effect on the CSR activity of AID, whereas all double and triple SHM in the presence of the DNA deamination can be also mutants showed severely reduced CSR activities. Collectively, all explained by defects in other functions such as inter- of the single mutants, especially at the residues between H48 and action. In addition, CSR and SHM activities of many AID N51, showed striking discordance between their DNA deami- mutants were affected independently, suggesting that AID may nation and CSR activities. The fact that CSR activity can be employ different mechanisms for CSR and SHM in consistence found in the N51A mutant without DNA deamination activity with reports (14–16). suggests that DNA deamination is not required for CSR. AID is well known to induce mutations in artificial target Titration of DNA Deamination and CSR Activities in B Cells. Because in nonlymphoid cells (18–20). We assessed the SHM our findings were quite unexpected from the assumption that dC activity of AID mutants by using a murine fibroblast cell line to dU deamination by AID initiates both CSR and SHM, we with an artificial GFP gene construct carrying a premature stop considered several possible explanations. First we had to exclude codon (20). This GFP reversion assay showed that D45A, R50A, any experimental bias by the comparison of the activities of AID

and N51A all had relatively weak SHM activity compared with proteins from different expression systems (one synthesized in IMMUNOLOGY wtAID, at comparable protein amounts (Fig. S4). Seven days vitro and the other in mammalian B-cells for the deamination

Shivarov et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15867 Downloaded by guest on September 29, 2021 Fig. 2. CSR activities of AID mutants. (A) The N51A mutant of mouse AID showed a significant level of CSR. Representative FACS plots show the level of CSR as assessed by the percentage of IgG1ϩ cells among the infected AIDϪ/Ϫ spleen cells (GFPϩ). The level of expression of AID for all infections was comparable is shown by GFPϩ percentages in total lymphocyte gate. (B) RT-PCR determination of the level of ␥1 switch transcripts from the same infected cells shown above confirmed the CSR activity of the mutant AID. (C) Summary of the CSR activity of all of the tested mutants as determined by FACS analysis. Bars above columns represent mean Ϯ SD. More than three independent experiments were performed. (D) SHM activity of AID mutants. The point mutation frequency was determined by sequencing a GFP construct from infectants of each AID mutant. The genomic DNA used for this analysis was pooled from three independent experiments. (E) Comparison of the ssDNA deamination, CSR, and SHM activities of all of the tested mutants relative to WT AID (100%).

activity and CSR assay, respectively). For example, there may be nation activity. In an independent experiment, we assessed the B cell factors that stimulate DNA deamination activity. To deamination activity of wtAID-ER at 4-OHT concentrations address these possibilities, it appeared essential to titrate pre- that give rise to the CSR activity equivalent to N51A (Ϸ20% cisely the CSR activity of the wtAID and its corresponding wtAID) (Fig. 3 C and D). Obviously, the DNA deamination deamination activity by using AID proteins in B cells. We activity of wtAID-ER was readily detectable at these 4-OHT assessed the CSR and DNA deamination activities of wtAID, concentrations (Fig. 3 E and F). The results confirmed that N51A, and KSS fused to human estrogen receptor (ER) by N51A has no DNA deamination activity yet catalyzes CSR, titration of 4-hydroxytamoxifen concentration (4-OHT) as de- indicating that DNA deamination is not required for CSR. scribed (12). CSR activity of both wtAID-ER and N51A-ER showed a clear dose dependency on 4-OHT concentration (Fig. Dissociation of DNA Deamination and Physiological Function in APO- 3 A and E). The maximum level of CSR activity we observed for BEC1. APOBEC1, another member of the C deaminase family, N51A under these conditions (48 h after 4-OHT addition) was has DNA deamination activity in vitro, although it is well 20% of wtAID-ER. Although we were able to demonstrate a established to be a RNA editing enzyme of ApoB 100 mRNA significant level of dose-dependent DNA deamination activity (21). Because N51 in AID is homologous to N57 in APOBEC1 for wtAID-ER, no deamination activity was detected for (Fig. S7), we examined DNA deamination and RNA editing N51A-ER even at saturating 4-OHT concentrations for CSR activities of the N57A mutant of APOBEC1. As shown in Fig. 4 (Fig. 3 B and E). KSS-ER had neither CSR nor DNA deami- A and B, N57A almost lost DNA deamination activity even at the

15868 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806641105 Shivarov et al. Downloaded by guest on September 29, 2021 4-OHT [nM] A 020100400

1 0.7 19.3 20.9 21.1

IgG 73 48 43 41.9

WT-ER

0.3 0.2 0.2 0.3 64.5 57.1 61.9 67

KSS-ER

0.4 1 3.6 5 KSS-ER Mock 78 78 73.7 70 WT-ER N51A-ER ER

N51A-ER tubulin GFP

4-OHT [nM] B 0 20 50 80 100 150 200 400 1000 KSS N51A Wild-type KSS Wild-type N51A Wild-type KSS N51A KSS N51A KSS Wild-type KSS N51A Wild-type KSS N51A KSS N51A Wild-type KSS N51A Oligo only b Wild-type Wild-type N51A Wild-type 26 13 1200150026 00 35 00 42 00 42 0 0 39 00 48 00 41 00 ref % cleavage

C 4-OHT [nM] D 4-OHT [nM] 02.55 7.5 10 02.557.510 100

0.9 3.35 6.5 6.8 8.7 66.4 63.5 58.4 58.3 55.9 b 1 Wild-type KSS N51A KSS N51A Wild-type KSS Wild-type KSS Wild-type KSS N51A KSS N51A Mock Wild-type N51A N51A Wild-type Oligo only

IgG 26 13 1.3 0 0 5.0 0 0 9.9 0 0 10.4 0 0 13.5 0 0 27.4 0 0 0 ref GFP % cleavage

E 120 120 F 120 120 WT-ER 100 100 100 100

80 80 80 80

60 60 60 60 WT-ER CSR [%] CSR [%] 40 40 40 40

20 N51A-ER 20 DEAMINATION [%] 20 20 DEAMINATION [%] DEAMINATION

0 0 0 0 0 50 100 150 200 250 300 350 400 450 022.5 5 7.5 10 00 4-OHT [nM] 4-OHT [nM]

Fig. 3. Determination of CSR and ssDNA deamination activities of ER-tagged wtAID and its mutants (KSS and N51A). (A) FACS plots after titration of 4-OHT. One day after retroviral infection and stimulation with LPS and IL-4, 4-OHT at increasing concentrations was added to each culture and the percentage of IgG1ϩ cells was determined 48 h later. Western blots represent the protein amounts after addition of 100 nM 4-OHT. (B) Polyacrilamide gel electrophoresis for determination of the DNA deamination activity of whole-cell lysates from the same experimental set used for CSR assay. Protein was extracted from cells incubated with 4-OHT at indicated concentrations by Dounce homogenization in PBS containing 10 ␮M ZnCl2 and 1% Nonidet P-40. Deamination assays were performed as described in Fig. 1. Percent cleavage was calculated by using densitometry. (C) FACS plots from an independent set of experiment for determination of wtAID-ER CSR activity at lower 4-OHT concentrations. (D) Deamination activity for wtAID-ER, KSS-ER, and N51A-ER determined in the same experiment as C. (E) Curve-fitted graph describing the relative activities of wtAID-ER (solid symbols) and N51A-ER (open symbols) based on the experiment described in A and B after subtraction of the levels at 0 nM 4-OHT. (F) Curve-fitted graph describing the relative activities of wtAID-ER based on the experiment described in C and D after subtraction of the levels at 0 nM 4-OHT. The activities of N51A-ER at 200 nM 4-OHT are taken from E.

enzyme to substrate ratio of 7.5. Nonetheless, N57A converted molecular mechanism underlying the anti-HIV function of the specific C in an ApoB 100 mRNA fragment into U with APOBEC3G is still unknown (24). It is still completely uncertain Ϸ20% efficiency of the WT APOBEC1 (Fig. 4C). This finding whether the DNA deamination activity of C deaminase family further supports our notion that DNA deamination activity of members, including AID, APOBEC1, and APOBEC3G, con- C deaminase does not necessarily represent its physiological tributes to their physiological functions. function. Finally, why is the DNA deamination activity preferentially Overexpressed APOBEC3G, which helps protect cells from lost whereas the CSR activity is relatively intact in the mutants HIV infection, deaminates C on cDNAs of the HIV clustered between G48 and N51? Because they have the catalytic genome (22). However, this DNA deamination function is not activity, a likely explanation is that the mutations in the G48-N51 required for the protection of host cells from HIV infection (23). region cause defect in a step before catalysis, such as binding to

One possible interpretation is that the DNA deamination activ- ssDNA, which may not be required for CSR. However, it is IMMUNOLOGY ity of APOBEC3G emerges when it is overexpressed. The difficult to pin point the affected step because there may be

Shivarov et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15869 Downloaded by guest on September 29, 2021 using wheat germ cell extracts (Cell Free Science) according to the manufac- A turer’s recommendations with minor modifications. In vitro translation was performed for 28 h at 17°C with a supplement of 10 ␮M ZnCl2. At the end of b AID APOBEC1APOBEC1(N57A) No AID synthesis, AID concentrations in wheat germ extracts were measured as 26 described in this paragraph. In vitro-synthesized wtAID protein was partially 13 purified from the wheat germ extract by using Niϩ beads (Qiagen) according % cleavage 82 36 1 0 to the manufacturer’s protocols. The purified wtAID protein and 1 ␮g control mouse IgG antibody were run on 15% SDS polyacrylamide gel electrophoresis anti-His (PAGE), followed by Coomassie brilliant blue staining. The amount of wtAID (25 kDa) was estimated by comparison with that of the light chain of the antibody (333 ng) by using densitometry. The amounts of AID mutants in 90 B wheat germ extracts were determined by comparison with known amounts of 80 AID 70 the purified wtAID, described above, with densitometry of Western blots by 60 using an anti-His monoclonal antibody. 50 40 APOBEC1 Deamination Assay. The DNA Glycosylase (UDG)-coupled deamination 30 assay has been described (10). Alexa-flour 680-labeled single-stranded oligonu- (% cleavage) 20 cleotide (Alexa-5Ј-TTTTTTTTTTTAGCGTTTTTTTTTTT-3Ј) was purchased from Ja-

Deamination activity 10 APOBEC1(N57A) pan BioService. Briefly, 0.1 pmol of labeled substrate was incubated with AID in 0 ␮ 0 1.5 7.5 crude wheat-germ extract, 0.4 unit UDG, and 1 g RNase for 60 min at 37°C in PBS supplemented with 10 ␮M ZnCl in a volume of 20 ␮l. The reaction was followed Molar ratio of enzyme to substrate 2 by incubation at 95°C for 8 min after addition of NaOH to 150 mM to cleave the alkali-labile abasic site. Samples were electrophoresed on 15% denaturing acryl- C C to U Total clones p amide gels and visualized by using Odyssey (Li-Cor). Cleavage ratio was deter- at ( C6666) mined by band quantitation by using Odyssey. AID + ACF 0 90 - APOBEC1 + ACF 18 91 0.00002 Frequency and Sequences of Mutations in E. coli. The mouse AID and its mutant APOBEC1 + ACF 8 116 0.022 (N57A) cDNAs were cloned into pET 23d vector (Novagen). The mutation assay was performed by using E. coli strain BL21 transformed with the vector alone or Fig. 4. In vitro DNA deamination and RNA editing activities of APOBEC1 the expression construct. Fluctuation assay and mutation analysis of rpoB mutant. (A) Representative polyacrylamide gel electrophoresis analysis of gene were performed as described (5). Within the 132-bp region analyzed, we R deamination activity by AID, APOBEC1, and its mutant. The experimental detected mutations in 61 of 81, 81 of 84, 83 of 88, and 78 of 81 Rif colonies condition is the same as in Fig. 1A.(B) Titration of AID, APOBEC1 and its transformed with the vector control, AID, KSS, and N51A, respectively. mutant proteins for deamination activity. The experimental condition is the ؊ ؊ same as in Fig. 1B except for the molar ratio of enzyme to substrate. Each assay CSR Assay in AID / Spleen B Cells. CSR assay was performed as described (16). was repeated more than three times. (C) RNA editing RT-PCR was done as described (8). activities of AID, APOBEC1, and its mutant were determined by sequencing from two independent experiments. An RNA fragment (239 b)containing C at SHM Assay in NIH 3T3-pI Clone 19 Cells. SHM assay in mouse fibroblast nt 6,666 of human apoB100 cDNA was synthesized in vitro and incubated at harboring GFP with premature stop codon (28) as an artificial substrate for 30°C for 2 h with the indicated proteins synthesized in vitro. RNA was SHM was performed as described (16, 20). extracted, reverse-transcribed, and amplified by using PCR for cloning. p, frequency of mutations was compared with AIDϩACF (Fisher’s exact test). 4-OHT Titration for Determination of CSR and Deamination Activity. ER-tagged constructs were obtained by cloning of the ER cDNA into pMSCV-wtAID-ires- GFP, pMSCV-KSS-ires-GFP, and pMSCV-N51A-ires-GFP. One day after infection several steps in the DNA deamination reaction. AID has been of AIDϪ/Ϫ cells stimulated with LPS and IL-4, cells were pooled, divided equally shown to bind not only DNA but also RNA (25), although the to add different concentrations of 4-OHT, and assayed for CSR and DNA deami- interaction of AID alone with both kinds of nucleic acids in vitro nation. Percentages of switched IgG1ϩ cells were determined 48 h later. On the is not sequence specific (9, 26). According to the recent report, same day, whole-cell lysates were prepared through Dounce homogenization in ␮ the AID mutants (S38A and S43P) show a high level of PBS containing 10 M ZnCl2 and 1% Nonidet P-40. ssDNA deamination assay was performed as described above by using 3 ␮g whole cell lysate per reaction. deamination activity at two non-hot-spot motifs, GGC and CGC, where AID is relatively inactive (27). However, N51A does not RNA Editing Assay. cDNAs of rat APOBEC1 and its mutant with 9-His-tag at the show any ssDNA deamination on these sequences (data not C-terminus and human APOBEC1 complementation factor (ACF) were cloned shown). If DNA deamination is not required for CSR, the into pEU-MCS vector and proteins were synthesized by in vitro transcription alternative explanation for the function of AID, i.e. RNA and translation as described above. For in vitro apoB RNA editing activity, targeting, is likely, although the question will remain open until 50-␮l editing reactions containing 1 ng of in vitro transcribed human apoB100 the actual RNA target of AID is identified. RNA (nucleotides 6,541–6,780; GenBank NM 000384) and whole wheat germ extracts containing equal amounts of AID, APOBEC1, and its mutant, in Materials and Methods addition to lysates containing ACF, were incubated at 30°C for2hinPBSand Mice. AID knockout mice on a C57BL/6 background (8) were maintained in our ZnCl2 10 nM. Editing efficiency at C6666 of apoB RNA was evaluated by animal facility under specific pathogen-free conditions. Spleen cells were sequencing after RT-PCR and cloning. obtained from these animals at 2–3 months of age. All mouse protocols were approved by the Institute of Laboratory Animals, Faculty of Medicine, Kyoto ACKNOWLEDGMENTS. We thank A. Kawamura and H. Hijikata for technical University (Kyoto, Japan). support, H. Nagaoka, I. Okazaki, N.A. Begum, and T. Doi for reading the manuscript and providing critical comments, Y. Endo, T. Sawasaki, and K. Okawa for critical suggestions in protein preparation, and Y. Shiraki and T. AID Protein Preparation. cDNAs of mouse AID and its various mutants with Nishikawa for technical assistance in preparing the manuscript. This work was 9-His-tag at C-termini were cloned into pEU-MCS vector for in vitro transcrip- supported by Ministry of Education, Culture, Sports, Science, and Technology tion and translation. AID and its mutant proteins were synthesized in vitro by of Japan Grant-in-Aid 17002015 for Specially Promoted Research.

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