Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in cis- and trans -Switching This information is current as of September 26, 2021. Jeroen E. J. Guikema, Carol E. Schrader, Niek G. J. Leus, Anna Ucher, Erin K. Linehan, Uwe Werling, Winfried Edelmann and Janet Stavnezer J Immunol 2008; 181:8450-8459; ; doi: 10.4049/jimmunol.181.12.8450 Downloaded from http://www.jimmunol.org/content/181/12/8450 References This article cites 54 articles, 30 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/181/12/8450.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 26, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in cis- and trans-Switching1 Jeroen E. J. Guikema,* Carol E. Schrader,* Niek G. J. Leus,2* Anna Ucher,* Erin K. Linehan,* Uwe Werling,† Winfried Edelmann,† and Janet Stavnezer3* When B cells are activated after immunization or infection, they exchange the gene encoding the Ig H chain C region by class switch recombination (CSR). CSR generally occurs by an intrachromosomal deletional recombination within switch (S) region sequences. However, ϳ10% of CSR events occur between chromosome homologs (trans- or interallele CSR), suggesting that the homologous chromosomes are aligned during CSR. Because the Mut S homolog 4 (Msh4) and Msh5 bind to Holliday junctions and are required for homologous recombination during meiosis in germ cells, we hypothesized these proteins might be involved Downloaded from in trans-chromosomal CSR (trans-CSR). Indeed, Msh4-Msh5 has recently been suggested to have a role in CSR. However, we find a large variety of alternative splice variants of Msh5 mRNA in splenic B cells rather than the full-length form found in testis. Most of these mRNAs are unlikely to be stable, suggesting that Msh5 might not be functional. Furthermore, we find that msh5 nul- lizygous B cells undergo CSR normally, have unaltered levels of trans-CSR, normal levels of DNA breaks in the S␮ region, and normal S-S junctions. We also show that the S-S junctions from cis- and trans-CSR events have similar lengths of junctional microhomology, suggesting trans-CSR occurs by nonhomologous end joining as does intrachromosome (cis)-CSR. From these http://www.jimmunol.org/ data, we conclude that Msh5 does not participate in CSR. The Journal of Immunology, 2008, 181: 8450–8459. lass switch recombination (CSR)4 is a unique type of pair enzyme uracil DNA glycosylase, and others are recognized by recombination that occurs in B cells upon appropriate the mismatch repair (MMR) proteins Mut S homolog (Msh)2- C activation. It results in a change from IgM and IgD ex- Msh6 (1–7). Additional base excision repair enzymes, apurinic/ pression by naive B cells to expression of IgG, IgE, or IgA. This apyrimidinic endonucleases 1 and 2, and several MMR enzymes switch changes CH, and improves the ability of the Ab to eliminate are required to generate the dsDNA breaks (DSBs) in the S regions the pathogen. It generally occurs by an intrachromosomal dele- that are required for CSR (8, 9). Deficiencies in the MMR proteins by guest on September 26, 2021 tional recombination between switch (S) region sequences located Msh2, Msh6, Mlh1, Pms2, and Exo1 reduce CSR by 2- to 5-fold ␦ upstream of each CH gene, except C . The CH genes are arranged (10–14). in the following order: C␮,C␦,C␥3, C␥1, C␥2b, C␥2a, C␧, and Msh5 is one of the Msh that are specialized for recognizing ␣ ␮ C , with the C gene being closest to the recombined VDJ gene aberrant DNA duplexes. However, Msh5 and its partner Msh4 do segment. CSR is initiated by activation-induced cytidine deami- not perform mismatch repair, but instead are essential for meiosis, nase (AID), which converts dC bases in S regions to dU bases, and mice deficient in either protein are sterile (15, 16). Msh5 does some of which are subsequently excised by the base excision re- not homodimerize, and its only known partner is Msh4 (17). Msh4-Msh5 forms a sliding clamp that binds to Holliday junctions in vitro (18), and during meiosis Msh4 is distributed along the *Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA synaptonemal complex early in prophase I, from the leptonema 01655; and †Department of Cell Biology, Albert Einstein Medical College, Bronx, stage until pachynema (16). Although meiotic chromosomes in NY 10461 msh4Ϫ/Ϫ or msh5Ϫ/Ϫ mice begin to form synaptonemal com- Received for publication April 18, 2008. Accepted for publication October 11, 2008. plexes, they do not condense correctly, and show abnormally high The costs of publication of this article were defrayed in part by the payment of page levels of association with Rad51. Rad51 association suggests that charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. meiosis is initiated and that DSBs and ssDNA breaks (SSB) are 1 This work was supported by National Institutes of Health Grants AI65639 (to generated as normal, but recombination between homologs fails to C.E.S.) and AI23283 (to J.S.), by the Irvington Institute Fellowship Program of the proceed normally. Deletion of either the msh4 or msh5 gene results Cancer Research Institute (to J.E.J.G.), and by CA76329 (to W.E.). in increased aberrant pairing between nonhomologous chromo- 2 Current address: Department of Medicine, University Medical Center Groningen, somes and greatly decreased pairing between homologs during Groningen, The Netherlands. meiosis in both spermatocytes and oocytes (15, 16). Taken to- 3 Address correspondence and reprint requests to Dr. Janet Stavnezer, Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, Uni- gether, the data suggest that during meiosis Msh4-Msh5 binds Hol- versity of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA liday junctions and monitors DNA sequences to ensure that 01655-0122. E-mail address: [email protected] recombination occurs between homologs. An additional het- 4 Abbreviations used in this paper: CSR, class switch recombination; AID, activation- erodimer, Mlh1-Mlh3, is also required for fertility and is thought induced cytidine deaminase; cis-CSR, intrachromosome-CSR; DSB, dsDNA break; KO, knockout; LM-PCR, ligation-mediated PCR; MMR, mismatch repair; Msh, Mut to bind and stabilize Msh4-Msh5 bound at Holliday structures S Homolog; S, switch; SSB, ssDNA break; TR, tandem repeat; trans-CSR, trans- (19–22). chromosomal CSR; WT, wild type. Msh4 and Msh5 mRNAs have been detected in several tissues Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 that do not contain cells undergoing meiosis, but whether they www.jimmunol.org The Journal of Immunology 8451 have a role in these tissues is unknown. Recently, Sekine et al. (23) FITC (AF6-78; BD Pharmingen). CFSE fluorescence and Ab staining were reported that Msh5 contributes to CSR. They showed that MRL/lpr acquired on a LSR flow cytometer (BD Biosciences) and analyzed using mice congenic for a 13-megabase segment from C57BL/6 (H-2b/b) the FlowJo software package (Tree Star). that includes the msh5 gene and the entire MHC locus have 5% of Western blotting the level of Msh5 mRNA in their spleens as do spleens from MRL/ k/k Cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, lpr H-2 mice. They found that these congenic mice have low 0.5% deoxycholate, 0.1% SDS, 1.0% Nonidet P-40, 50 mM Tris-Cl (pH levels of serum IgG3. They also found that splenic B cells from 7.5), Complete protease, and phosphatase inhibitor mixture; Roche) and msh4 or msh5 knockout (KO) mice had altered S␮-S␥3 and S␮-S␣ subjected to three freeze-thaw cycles to obtain whole-cell protein extracts. junctions relative to wild-type (WT) littermates. From additional Protein content was determined using Bradford assay (Bio-Rad). Equal amounts (25 ␮g) of proteins were electrophoresed on 8% SDS-polyacryl- analyses of patients with various single-nucleotide mutations amide gels, and blotted onto Immobilon-P polyvinylidene fluoride mem- within the msh5 gene, they concluded that Msh5 has a role in CSR, branes (Millipore). Immunoblotting was performed using rabbit anti-Msh5, although the role was not defined. followed by goat anti-rabbit HRP (Santa Cruz Biotechnology) and en- Most switch recombination occurs by an intrachromosomal de- hanced chemiluminescent substrate (Pierce). letion event, although recently it was demonstrated that 7–17% of Genomic DNA preparation and linker ligation-mediated PCR the CSR events occur between the two homologous chromosomes (LM-PCR) (24–26). Because trans-chromosomal CSR would require align- After culture for 2 days, viable cells were isolated by Ficoll/Hypaque gra- ment of the two chromosome homologs, we hypothesized that dients (r ϭ 1.09) or Lympholyte (Cedarlane Laboratories) centrifugation.