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Jimmunol.0901240.Full.Pdf A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Gene 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 Supplementary http://www.jimmunol.org/content/suppl/2009/07/07/jimmunol.090124 Material 0.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 genes undergo somatic hypermutation (SHM)2 at serves as a scaffold that mediates protein-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 adenine (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 mutagenesis (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 gene expression 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.
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