A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Hypermutation

This information is current as Keiji Masuda, Rika Ouchida, Yingqian Li, Xiang Gao, of September 24, 2021. Hiromi Mori and Ji-Yang Wang J Immunol published online 8 July 2009 http://www.jimmunol.org/content/early/2009/07/08/jimmuno l.0901240 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 8, 2009, doi:10.4049/jimmunol.0901240 The Journal of Immunology

A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Gene Hypermutation

Keiji Masuda,* Rika Ouchida,* Yingqian Li,†* Xiang Gao,† Hiromi Mori,* and Ji-Yang Wang1*

REV1 is a deoxycytidyl transferase that catalyzes the incorporation of deoxycytidines opposite deoxyguanines and abasic sites. To explore the role of its catalytic activity in Ig gene hypermutation in mammalian cells, we have generated mice expressing a catalytically inactive REV1 (REV1AA). REV1AA mice developed normally and were fertile on a pure C57BL/6 genetic back- ground. B and T cell development and maturation were not affected, and REV1AA B cells underwent normal activation and class switch recombination. Analysis of Ig gene hypermutation in REV1AA mice revealed a great decrease of C to G and G to C transversions, consistent with the disruption of its deoxycytidyl transferase activity. Intriguingly, REV1AA mice also exhibited a significant reduction of C to T and G to A transitions. Moreover, each type of nucleotide substitutions at A:T base pairs was

uniformly reduced in REV1AA mice, a phenotype similar to that observed in mice haploinsufficient for Polh. These results reveal Downloaded from an unexpected role for REV1 in the generation of C:G transitions and A:T mutations and suggest that REV1 is involved in multiple mutagenic pathways through functional interaction with other polymerases during the hypermutation process. The Journal of Immunology, 2009, 183: 0000–0000.

he Ig undergo somatic hypermutation (SHM)2 at serves as a scaffold that mediates -protein interactions. De-

both C:G and A:T base pairs in the germinal centers (GC) letion of the BRCT domain of REV1 in ES cells resulted in in- http://www.jimmunol.org/ T B cells (1). SHM is initiated by activation-induced cyti- creased sensitivity to DNA-damaging agents, indicating that this dine deaminase (AID), which catalyzes the deamination of cyto- domain is involved in tolerance to DNA damage (19). The SHM of sine (C) to uracil (U) and generates a U:G mismatch on DNA (2, Ig genes in BRCT-mutant mice was reportedly normal, both in 3). Accumulating evidence suggests that mutations are introduced terms of mutation frequency and patterns (19). The polymerase during replication and repair of the U:G mismatch (1). Direct rep- domain is essential for the deoxycytidyl transferase activity and lication of the U:G mismatch would generate C to T and G to A mutations of the highly conserved aspartate and glutamate residues transitions because U normally pairs with (A). In addition, to alanines completely abolished the catalytic activity of human U can be excised by the uracil DNA glycosylase (UNG), and rep- and mouse REV1 (15, 18). The C-terminal region of REV1 has

lication of the resulting abasic site by the low-fidelity DNA poly- been shown to mediate the interaction with Y-family polymerases, by guest on September 24, 2021 merases would generate both transitions and transversions at C:G including POLK, POLI, and POLH (18, 20). REV1-deficient mice base pairs. The U:G mismatch and the abasic site can also be exhibited reduced body sizes and were not fertile after backcross- processed by a mismatch repair-like and a base excision repair-like ing for two generations to C57BL/6 mice (21). Analysis of Ig gene pathway, respectively, resulting in the generation of mutations at SHM in these mice revealed an absence of C to G and a reduction undamaged A:T base pairs (4–7). Studies thus far suggest that of G to C transversions and a compensatory increase of C to A, A DNA polymerase ␩ (POLH) is an essential enzyme for the induc- to T, and T to C mutations (21). These observations have led to the tion of A:T mutations in both pathways (8–13). conclusion that REV1 participates in Ig gene SHM primarily by REV1 is a deoxycytidyl transferase that preferentially incorpo- inserting C opposite abasic sites generated by UNG. rates deoxycytidines opposite deoxyguanines and abasic sites (14– To investigate the role of the catalytic activity of REV1 in SHM 18). REV1 contains a BRCA1 C-terminal (BRCT) domain in the in mammalian cells, we have generated Rev1 knockin mice ex- N terminus, a central polymerase domain, and a Y-family poly- pressing a catalytically inactive REV1 (REV1AA). REV1AA mice merase contacting region in the C terminus. The BRCT domain exhibited a great reduction of not only C to G and G to C trans- versions but also C to T and G to A transitions and A:T mutations.

*Laboratory for Immune Diversity, Research Center for Allergy and Immunology, Because REV1 itself does not possess the catalytic property to RIKEN Yokohama Institute, Yokohama, Japan; and †Model Animal Research Center, directly introduce C:G transitions and A:T mutations, these obser- Ministry of Education Key Laboratory of Model Animal for Disease Research, Med- vations suggest that REV1 regulates the induction of these muta- ical School of Nanjing University, Nanjing, China tions through functional interaction with other polymerases during Received for publication April 17, 2009. Accepted for publication June 2, 2009. Ig gene hypermutation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 Address correspondence and reprint requests to Dr. Ji-Yang Wang, Laboratory for Generation of mice expressing REV1AA Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. E-mail address: The highly conserved aspartate and glutamate residues encoded by exon 10 [email protected] were mutated to alanines by site-directed (Takara Bio). A fragment containing the mutated exon 10 was used as the 5Ј-arm of the 2 Abbreviations used in this paper: SHM, somatic hypermutation; BRCT, BRCA1 C-terminal; GC, germinal center; MMS, methyl methanesulfonate; POLH, polymer- targeting vector. Transfection and selection of the C57BL/6-derived ase ␩; REV1AA, catalytically inactive REV1; UNG, uracil DNA glycosylase; WT, Bruce4 ES cells were performed as described previously (22). Homologous wild type. recombinants were identified by long-range genomic PCR using primers s1 (5Ј-GCTTTGTCTTTGGGGGATGA-3Ј) and neos (5Ј-TCGCCTTCTATC Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 GCCTTCTT-3Ј) for 5Ј and as1 (5Ј-TCAGCCACATCCACATACCC-3Ј)

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901240 2 REV1 INVOLVED IN C:G TRANSITIONS AND A:T MUTATIONS Downloaded from http://www.jimmunol.org/

FIGURE 1. Generation of REV1AA mice. A, Targeting strategy. The targeting vector was designed to replace exon 10 (E10) with a mutated E10 (indicated by an “x”) in which the highly conserved aspartate and glutamate residues were substituted with alanines. The positions of PCR primers (s1, as1, s2, as2, s3, as3, neos, and neoas) are indicated. B, Long-range PCR analysis to detect the targeted allele, using primers s1 and neos for the 5Ј-arm and as1 and neoas for the 3Ј-arm. The predicted sizes for the 5Ј- and 3Ј-PCR were 4.8- and 6.3-kb, respectively. M, size markers. C, The neo gene was deleted

by crossing the mutant mice with CAG-Cre transgenic mice, leaving a single ϳ35-bp loxP site in the intron between exons 10 and 11. Mouse genotypes by guest on September 24, 2021 were screened by PCR using primers s2 and as2 flanking the loxP site. WT and targeted alleles gave rise to 364- and 400-bp bands, respectively. M, size markers. D, Northern blot analysis for Rev1 in WT and REV1AA spleen B cells (upper panel). A 1.6-kb Rev1 cDNA fragment encom- passing exons 12–22 (corresponding to cDNA sequence 2101ϳ3695) was used as a probe. Lower panel, ␤-actin expression as a control. E, A genomic fragment containing exon 10 was amplified using primers s3 and as3 from WT, heterozygous, and homozygous mice and analyzed by direct sequencing. and neoas (5Ј-ATAGCCGAATAGCCTCTCCA-3Ј) for 3Ј homology re- were sorted from spleen of each mouse, and the genomic DNA was ex- gions. PCR was performed at 95°C for 2 min, followed by 30 cycles of tracted. The JH4 intronic region was amplified and sequenced as described amplification at 95°C for 10 s, 57°C for 20 s, and 68°C for 10 min (for 5Ј) previously (23). Only unique sequences were analyzed in each mouse. or 95°C for 10 s, 63°C for 20 s, and 68°C for 13 min (for 3Ј) using LA-Taq polymerase (Takara Bio). Chimeric mice were bred with C57BL/6 mice to Sensitivity to DNA damaging agents obtain heterozygotes, which were then crossed with CAG-Cre transgenic ϫ mice to delete the neo gene. The neo-deleted heterozygous mice were bred Purified spleen B cells were seeded in 12-well plates in duplicate at 5 5 ␮ to obtain homozygous REV1AA mice. Mouse genotypes were determined 10 /ml (1 ml/well) in the presence of 10 g/ml LPS. The cells were then by PCR using primers s2 (5Ј-CGATGTTAATGTCACAATGG-3Ј) and as2 exposed to different doses of methyl methanesulfonate (MMS) or cisplatin (5Ј-CAACGTGTAGAAAGCTAAGC-3Ј) at 95°C for 2 min, followed by or irradiated with UV light and cultured for 2 days. The cells were then FITC 30 cycles of amplification at 95°C for 10 s, 56°C for 20 s, and 72°C for 1 stained with Annexin V and propidium iodide (PI) to detect apoptotic and dead cells, respectively. The percentages of live cells (Annexin min using Taq polymerase (Toyobo). To further verify the introduced mu- Ϫ Ϫ tations, a genomic fragment containing exon 10 was amplified using prim- V PI ) were determined by FACS. ers s3 (5Ј-ATGTGTTGGTGTGCTGGTGT-3Ј) and as3 (5Ј-ATTTGGG AGAACTTTAGGTC-3Ј) and directly sequenced. PCR was performed at Results 95°C for 2 min, followed by 30 cycles of amplification at 95°C for 10 s, Generation of REV1AA mice 56°C for 20 s, and 68°C for 1 min using KOD plus polymerase (Toyobo). Mice were maintained in specific pathogen-free conditions, and all exper- We mutated the highly conserved aspartate and glutamate residues iments were approved by the Animal Facility Committee of RIKEN Yoko- encoded by exon 10 to alanines by gene targeting (Fig. 1, A–C). hama Institute (permission no. 20-025). Northern blot analysis revealed a similar transcript level of Rev1 FACS analysis, B cell purification, proliferation, and class gene in WT and REV1AA B cells (Fig. 1D), indicating that the switch assay targeted mutations did not affect Rev1 gene transcription. Se- quence analysis of a fragment containing exon 10 further verified These experiments were performed as described previously (23). the designated mutations (Fig. 1E). Attempts to detect WT and SHM of Ig genes mutant REV1 protein by immunoblot have been unsuccessful due Five REV1AA mice and four wild-type (WT) littermates were injected i.p. to the poor specificity of the available Abs. However, it has been with 100 ␮g of 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken ␥-glob- shown previously that the corresponding mutations in human ulin precipitated with alum. Two weeks later, B220ϩPNAhigh GC B cells REV1 did not affect the global protein structure, and the mutant The Journal of Immunology 3

FIGURE 2. Normal B cell devel- opment, maturation, and activation in REV1AA mice. A, FACS profiles of gated B220ϩ cells in the BM and spleen. B, FACS profiles of thymo- cytes and splenocytes. C, Prolifera- tive responses of purified spleen B cells to various stimuli. D, Class switch from IgM to IgG1 in WT and REV1AA B cells after in vitro stim- Downloaded from ulation with LPS ϩ IL-4 (upper pan- els) or CD40L ϩ IL-4 (lower panels) for 3 days. Representative results of three independent experiments are shown. http://www.jimmunol.org/ by guest on September 24, 2021

REV1 could be successfully purified like the WT protein (15). Reduced frequency of C:G transitions and A:T mutations in These observations collectively suggest that REV1AA mice ex- REV1AA mice press normal levels of REV1AA. To address the role of the catalytic activity of REV1 in Ig gene Normal B cell development, maturation, and activation in SHM, we analyzed the JH4 intronic region of GC B cells isolated REV1AA mice from spleens of immunized mice. We have analyzed four WT and five REV1AA mice, and the results are summarized in Table I (see REV1AA mice were derived from Bruce4 ES cells and were there- supplemental Table I for detailed results of individual WT and fore on a pure C57BL/6 genetic background. The homozygous REV1AA mice).3 Consistent with our previous studies (24), the mice were born at the expected ratio and developed normally with ϳ overall mutation frequency in the JH4 intronic region was 1% in no obvious abnormalities by appearance. FACS analysis of BM WT mice with roughly half of the mutations occurring at C:G and cells of WT and REV1AA mice revealed no obvious differences in ϩ ϩ Ϫ ϩ Ϫ A:T. The overall mutation frequency was reduced by 35% in the percentages of B220 CD43 IgM progenitor, B220 CD43 REV1AA mice (0.651 vs 0.996% in WT mice). Mutations at C:G Ϫ dull ϩ high ϩ IgM precursor, B220 IgM immature, and B220 IgM re- and A:T were decreased by 37 and 33%, respectively, compared circulating B cells (Fig. 2A, upper and middle panels). The pro- with WT mice (Table I). To determine more precisely how SHM portion of CD23highCD21dull follicular and CD23dullCD21high marginal zone B cells in the spleen was also similar between WT and REV1AA mice (Fig. 2A, lower panels). T cell development and differentiation were not affected in REV1AA mice as judged by Table I. Mutation frequency in WT and REV1AA mice CD4 and CD8 profiles in the thymus and spleen (Fig. 2B). In addition, REV1AA B cells exhibited normal proliferative responses to anti- REV1AA IgM Abs, CD40L and LPS (Fig. 2C), and switched normally from JH4 Intron (509 bp) WT (four mice) (five mice)

IgM to IgG1 upon in vitro stimulation (Fig. 2D). B cell activation in No. of sequences 494 570 vivo was also normal as revealed by a similar representation of GC B Mutated sequences (%) 372 (75.3%) 400 (70.2%) cells in WT and REV1AA mice after immunization with a T-depen- Total length of mutated sequences 189,348 203,600 3 Total number of mutations 1,886 1,325 dent Ag (supplemental Fig. 1). These results collectively suggest that Overall mutation frequency (%) 0.996 0.651a B and T cell differentiation and activation were not affected by the Mutation frequency at C:G (%) 0.460 0.291 Mutation frequency at A:T (%) 0.536 0.360 disruption of the deoxycytidyl transferase activity of REV1. % mutation at C:G : A:T 46.2:53.8 44.7:55.3

a The values in bold type indicate significant differences from WT mice ( p Ͻ 0.05, 3 The online version of this article contains supplemental material. unpaired t test). 4 REV1 INVOLVED IN C:G TRANSITIONS AND A:T MUTATIONS

transversions (27). These observations are consistent with the cat- alytic property of REV1 and suggest that REV1 participates in Ig gene SHM by incorporating C opposite UNG-mediated abasic sites. However, unlike REV1-deficient mice in which C to G trans- versions were virtually absent while G to C transversions were reduced, we found a similar reduction of C to G and G to C trans- versions in REV1AA mice. It is unclear at this point why REV1- deficient but not REV1AA mice exhibit a strand-biased defect in C:G transversions. Both REV1-deficient and REV1AA mice exhibited a significant increase of C to A and a moderate increase of G to T transversions. These observations suggest that induction of these mutations not FIGURE 3. Absolute frequency of each type of nucleotide substitutions only does not require REV1 but also is not inhibited by the pres- -Significant differences between WT and ence of an inactive REV1. Therefore, these mutations are gener ,ء .in WT and REV1AA mice REV1AA mice (p Ͻ 0.05, unpaired t test). See supplemental Table II3 for ated by a REV1-independent pathway. The increased C to A and, detailed results of individual mice. to a lesser extent, G to T transversions are likely due to compen- satory activation of this mutagenic pathway in the absence of a functional REV1. was affected in REV1AA mice, we calculated the absolute fre- The reduction of C to T and G to A transitions and A:T muta- Downloaded from quency of each type of nucleotide substitutions. For this purpose, tions observed in REV1 AA mice is unexpected because REV1 we included data of WT mice from our previous studies (24), does not possess the catalytic property to directly introduce these allowing the analysis of a large number of each type of mutations mutations. According to the DNA deamination model of Ig gene for comparison. As expected, C to G and G to C transversions were SHM, C:G transitions can be generated by replication of either greatly decreased (75 and 71% reduction, respectively) in activation-induced deaminase-triggered U:G lesion or REV1AA mice, with a significant increase of C to A substitution

UNG-mediated abasic site (1). Replication of the U:G lesion is http://www.jimmunol.org/ (Fig. 3 and supplemental Table II).3 Intriguingly, C to T and G to thought to be conducted by replicative polymerases, and it is thus A transitions were also significantly decreased (47 and 49% re- unlikely that REV1 participates in this process. It is more likely duction, respectively) in REV1AA mice as compared with WT that REV1 is involved in the generation of C:G transitions during mice. Moreover, we found a uniform reduction of each type of replication of the abasic site. Mammalian REV1 has been shown to nucleotide substitutions at A:T base pairs in REV1AA mice (Fig. interact with Y-family polymerases, raising the possibility that 3 and supplemental Table II),3 a phenotype resembling that ob- ϩ Ϫ REV1 may regulate the generation of C:G transitions by recruiting served in Polh / mice (24). other polymerases. This possibility, however, is unlikely because Normal sensitivity to DNA damaging agents in REV1AA B cells the interaction of REV1 with Y-family polymerases is mediated by the C-terminal region of REV1, which is intact in REV1AA mice. by guest on September 24, 2021 REV1 is known to play a critical noncatalytic role in tolerance to A more likely possibility is that the REV1AA might be stuck or DNA damage in yeast and chicken DT40 cells presumably by stabilized at the abasic site, thereby preventing the switching be- interacting with other polymerases and coordinating their activities tween REV1 and other polymerases involved in the generation of during translesion DNA synthesis (25, 26). Consistently, WT and C:G transitions. A similar mechanism may account for the reduc- REV1AA B cells exhibited similar sensitivities to MMS, cisplatin, tion of A:T mutations in REV1AA mice. The induction of A:T and UV light (Fig. 4). In addition, cell cycle analysis revealed no mutations is highly dependent on POLH (8–13), and REV1AA differences in the relative proportion of cells at G , S, and G -M 1 2 might inhibit the access of POLH to the site of DNA lesions. We phases between WT and REV1AA B cells treated with these have recently found that Polh heterozygous mice exhibit a uniform agents (data not shown). These results demonstrate that REV1 cat- reduction of each type of nucleotide substitutions at A:T pairs (24). alytic activity per se is not essential for tolerance to DNA damage A similar uniform reduction of A:T mutations in REV1AA mice is in mammalian B cells. consistent with the hypothesis that POLH function is compromised in the presence of an inactive REV1. Regardless of the precise Discussion mechanism, our results suggest that REV1 and other polymerases, The great reduction of C to G and G to C transversions in including POLH, do not function independently, and the interac- REV1AA mice is in agreement with the results of REV1-deficient tion and switching between these polymerases are important for mice (21). In addition, REV1-deficient chicken DT40 cells recon- efficient induction of somatic mutations in Ig genes. Recently, stituted with a catalytically inactive human REV1 (D570A/ REV1-POLH interaction was shown to be required for the forma- E571A) also exhibited a dramatic reduction of C to G and G to C tion of REV1 foci and for suppression of spontaneous mutations in human cells (28). It would be interesting to generate mice express- ing a mutant REV1 that has normal catalytic activity but cannot interact with POLH and other Y-family polymerases. The reduction of C:G transitions and A:T mutations in REV1AA mice was not observed in REV1-deficient mice. If the reduction of these mutations is indeed due to the stabilization of REV1AA at the site of DNA lesions, it is not surprising that the absence of REV1 did not affect the generation of these mutations. FIGURE 4. Survival (percentage of live cells) of WT and REV1AA Conversely, the increased A to T and T to C substitutions were spleen B cells exposed to MMS, cisplatin, or UV. Mean Ϯ SD of duplicate observed in REV1-deficient but not REV1AA mice. In this case, wells are shown. The survival of untreated cells was set at 100%. Similar the absence of REV1 might have resulted in increased access of results were obtained in three independent experiments. POLH to the abasic site, leading to the increase of certain A:T The Journal of Immunology 5

mutations. The differences in the mutation patterns between 11. Martomo, S. A., W. W. Yang, R. P. Wersto, T. Ohkumo, Y. Kondo, M. Yokoi, REV1AA and REV1-deficient mice might also be due to the dif- C. Masutani, F. Hanaoka, and P. J. Gearhart. 2005. Different mutation signatures in DNA polymerase ␩- and MSH6-deficient mice suggest separate roles in an- ferent methods used to analyze SHM. We have analyzed the JH4 tibody diversification. Proc. Natl. Acad. Sci. USA 102: 8656–8661. intronic region of GC B cells isolated from immunized mice, and 12. Delbos, F., A. De Smet, A. Faili, S. Aoufouchi, J. C. Weill, and C. A. Reynaud. ␩ the mutations are induced within 2 wk by acute Ag stimulation. In 2005. Contribution of DNA polymerase to immunoglobulin gene hypermuta- ␭ tion in the mouse. J. Exp. Med. 201: 1191–1196. contrast, V 1 genes of memory B cells isolated from 3- to 5-mo- 13. Delbos, F., S. Aoufouchi, A. Faili, J. C. Weill, and C. A. Reynaud. 2007. DNA old mice were analyzed for Rev1-deficient mice, and these mem- polymerase ␩ is the sole contributor of A/T modifications during immunoglobulin ory B cells had likely undergone relatively long-term selection by gene hypermutation in the mouse. J. Exp. Med. 204: 17–23. 14. Lin, W., H. Xin, Y. Zhang, X. Wu, F. Yuan, and Z. Wang. 1999. The human endogenous Ags. Further studies are required to completely reveal REV1 gene codes for a DNA template-dependent dCMP transferase. Nucleic how REV1 participates in Ig gene SHM. In conclusion, our results Acids Res. 27: 4468–4475. suggest that REV1 is involved in multiple mutagenic pathways and 15. Masuda, Y., M. Takahashi, N. Tsunekuni, T. Minami, M. Sumii, K. Miyagawa, and K. Kamiya. 2001. Deoxycytidyl transferase activity of the human REV1 contributes to the generation of a significant portion of both C:G protein is closely associated with the conserved polymerase domain. J. Biol. and A:T mutations during Ig gene hypermutation. Chem. 276: 15051–15058. 16. Masuda, Y., and K. Kamiya. 2002. Biochemical properties of the human REV1 Acknowledgments protein. FEBS Lett. 520: 88–92. 17. Masuda, Y., M. Takahashi, S. Fukuda, M. Sumii, and K. Kamiya. 2002. Mech- We thank C. Stewart for providing the Bruce4 ES cells, Y. Masuda for anisms of dCMP transferase reactions catalyzed by mouse Rev1 protein. J. Biol. helpful discussions, the Animal Facility for breeding and maintaining the Chem. 277: 3040–3046. mice, and the FACS Laboratory for cell sorting. 18. Guo, C., P. L. Fischhaber, M. J. Luk-Paszyc, Y. Masuda, J. Zhou, K. Kamiya, C. Kisker, and E. C. Friedberg. 2003. Mouse Rev1 protein interacts with multiple Disclosures DNA polymerases involved in translesion DNA synthesis. EMBO J. 22: Downloaded from 6621–6630. The authors have no financial conflict of interest. 19. Jansen, J. G., A. Tsaalbi-Shtylik, P. Langerak, F. Calleja, C. M. Meijers, H. Jacobs, and N. de Wind. 2005. The BRCT domain of mammalian Rev1 is References involved in regulating DNA translesion synthesis. Nucleic Acids Res. 33: 1. Di Noia, J. M., and M. S. Neuberger. 2007. Molecular mechanisms of antibody 356–365. somatic hypermutation. Annu. Rev. Biochem. 76: 1–22. 20. Ohashi, E., Y. Murakumo, N. Kanjo, J. Akagi, C. Masutani, F. Hanaoka, and 2. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and H. Ohmori. 2004. Interaction of hREV1 with three human Y-family DNA poly- T. Honjo. 2000. Class switch recombination and hypermutation require activa- merases. Genes Cells 9: 523–531. http://www.jimmunol.org/ tion-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 21. Jansen, J. G., P. Langerak, A. Tsaalbi-Shtylik, P. van den Berk, H. Jacobs, and 102: 553–563. N. de Wind. 2006. Strand-biased defect in C/G transversions in hypermutating 3. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F. W. Alt. 2003. immunoglobulin genes in Rev1-deficient mice. J. Exp. Med. 203: 319–323. Transcription-targeted DNA deamination by the AID antibody diversification en- 22. Ouchida, R., S. Yamasaki, M. Hikida, K. Masuda, K. Kawamura, A. Wada, zyme. Nature 422: 726–730. S. Mochizuki, M. Tagawa, A. Sakamoto, M. Hatano, et al. 2008. A lysosomal 4. Rada, C., M. R. Ehrenstein, M. S. Neuberger, and C. Milstein. 1998. Hot spot protein negatively regulates surface T cell antigen receptor expression by pro- focusing of somatic hypermutation in MSH2-deficient mice suggests two stages moting CD3␨-chain degradation. Immunity 29: 33–43. of mutational targeting. Immunity 9: 135–141. 23. Masuda, K., R. Ouchida, M. Hikida, T. Kurosaki, M. Yokoi, C. Masutani, 5. Frey, S., B. Bertocci, F. Delbos, L. Quint, J. C. Weill, and C. A. Reynaud. 1998. M. Seki, R. D. Wood, F. Hanaoka, and J. O. Wang. 2007. DNA polymerases ␩ Mismatch repair deficiency interferes with the accumulation of mutations in and ␪ function in the same genetic pathway to generate mutations at A/T during chronically stimulated B cells and not with the hypermutation process. Immunity

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