Activation-Induced Cytidine Deaminase-Dependent DNA Breaks in Class Switch Recombination Occur during G1 Phase of the Cell Cycle and Depend upon This information is current as Mismatch Repair of September 27, 2021. Carol E. Schrader, Jeroen E. J. Guikema, Erin K. Linehan, Erik Selsing and Janet Stavnezer J Immunol 2007; 179:6064-6071; ; doi: 10.4049/jimmunol.179.9.6064 Downloaded from http://www.jimmunol.org/content/179/9/6064 References This article cites 51 articles, 21 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/179/9/6064.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 27, 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Activation-Induced Cytidine Deaminase-Dependent DNA Breaks in Class Switch Recombination Occur during G1 Phase of the Cell Cycle and Depend upon Mismatch Repair1 Carol E. Schrader,2* Jeroen E. J. Guikema,* Erin K. Linehan,* Erik Selsing,† and Janet Stavnezer2* Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs ␮ are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in S mainly in the G1 phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G1 phase cells express AID, UNG, and mismatch Downloaded from repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/S␮TR؊/؊ B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously. The Journal of Immunology, 2007, 179: 6064–6071. http://www.jimmunol.org/ ntibody class switch recombination (CSR)3 involves the Repair of the dU residues resulting from AID activity leads to replacement of the IgM C region gene (C␮) by a down- DNA breaks necessary for recombination. Uracil in DNA is re- A stream C region gene, e.g., C␥, C␣,orC␧, and can im- moved by uracil DNA glycosylase (UNG) and CSR is severely prove the efficacy of the immune response. The recombination reduced in mice that lack UNG and in patients with mutations in occurs via the formation of double-strand breaks (DSBs) in switch UNG (13, 14). UNG activity generates abasic sites that could be (S) region DNA located upstream of the C region genes, and the recognized and cleaved by AP endonucleases (APE) to create sin- joining of two different S regions by an end-joining mechanism, gle-strand breaks (SSBs), and recent data indicate that APE is im- resulting in deletion of the intervening DNA from the genome (1). portant for CSR (53). AID- and UNG-dependent DSBs have been CSR requires activation induced cytidine deaminase (AID), which detected in S regions, and the breaks occur preferentially at G:C bp by guest on September 27, 2021 converts cytosines in DNA to uracils (2–7). The target for AID is in AID hot-spot motifs, which is consistent with cleavage by APE ssDNA, which can be generated during transcription, and only at the nucleotide deaminated by AID (14–17). The current data transcriptionally active S regions undergo CSR (1, 4, 8–10). S support the conclusion that repair of the dU residues generates regions consist of tandem repeats (TR) that are unique to each SSBs, but SSBs must be converted to DSBs to excise the DNA isotype, although all contain numerous targets for AID: the hot- segment intervening between S␮ and the downstream S region. spot motif WRC/GYW, where W ϭ AorT,Rϭ GorA,andYϭ Currently, nothing is known about the mechanism by which C or T (6, 11, 12). The underlined C is determinated by AID. SSBs in S regions become DSBs. In this study, we test three hy- potheses regarding how DSBs are created during CSR. SSBs ini- tiated by AID activity could be converted into DSBs during rep- *Department of Molecular Genetics and Microbiology, Program in Immunology and lication, when the elongating DNA strand reaches a nick on the Virology, University of Massachusetts Medical School, Worcester, MA 01655; and template strand. However, ␥H2AX and Nbs-1, two proteins in- †Genetics Program and the Department of Pathology, Tufts University School of Medicine, Boston, MA 02111 volved in CSR, are found associated with the IgH locus during G /early S phase, but not during S/G M phase in splenic B cells Received for publication June 30, 2007. Accepted for publication August 22, 2007. 1 2 undergoing CSR (18). To address this issue, we analyzed the cell 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 cycle regulation of DSBs in S regions, with emphasis on separation with 18 U.S.C. Section 1734 solely to indicate this fact. of G1 phase from S phase cells. 1 This work was supported by National Institutes of Health Grants AI065639 (to Nucleotide excision repair (NER) could recognize a variety of C.E.S.), AI23283 and AI632026 (to J.S.), and by the Cancer Research Institute (to damage intermediates resulting from AID activity and could in- J.E.J.G.). troduce DNA breaks with its associated endonucleases Ercc1-Xe- 2 Address correspondence and reprint requests to Dr. Carol E. Schrader and Dr Janet Stavnezer, Department of Molecular Genetics and Microbiology, Program in Immu- roderma pigmentosum F (XPF) and XPG (19). A minor role for nology and Virology, University of Massachusetts Medical School, 55 Lake Avenue Ercc1 in CSR has been described (20), but it is not known whether North, Worcester, MA 01655; E-mail addresses: [email protected] or Ercc1-XPF acts during CSR in conjunction with the NER pathway [email protected] or independently as a structure-specific endonuclease. We tested 3 Abbreviations used in this paper: CSR, class switch recombination; DSB, double- strand break; AID, activation-induced cytidine deaminase; TR, tandem repeat; UNG, whether XPA, which is essential for NER, is involved in CSR. uracil DNA glycosylase; APE, AP endonuclease; SSB, single-strand break; NER, Nicks sufficiently close (Ͻ5 bp apart) on opposite strands can nucleotide excision repair; MMR, mismatch repair; WT, wild type; LM-PCR, liga- form DSBs spontaneously, as shown by experiments in which the tion-mediated PCR; XPF, Xeroderma pigmentosum F. restriction enzyme I-Sce1 can produce a chromosomal DSB (21). Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 However, distal SSBs will not lead to DSBs, due to stability of the www.jimmunol.org The Journal of Immunology 6065 DNA duplex, unless the DNA ends are processed. AID is known to deaminate dC nucleotides on both the transcribed and nontran- scribed strands (22, 23), and the density of AID hot spots occurring in the S region TRs could lead to nicks on opposite strands in close proximity to each other. However, deletion of the S␮TR region results in only a modest (2-fold) reduction in CSR (24). CSR can occur upstream of, as well as within, the S␮TR region (25, 26), although recombination outside of the S␮TRs requires the DNA mismatch repair (MMR) pathway (26, 27). In fact, CSR is nearly ablated in S␮TRϪ/Ϫ B cells that also lack the MMR proteins Msh2 or Mlh1 (28) (J. Eccleston, C. E. Schrader, J. Stavnezer, and E. Selsing, manuscript in preparation). Because MMR is required for recombination outside the S␮TR region where AID hot spots are relatively infrequent, we have proposed that MMR is needed to convert SSBs into DSBs when the single-strand nicks are too far apart to form spontaneous DSBs (29). This hypothesis is consistent with the finding that CSR does not require MMR, but is reduced 2- to 5-fold in MMR-deficient B cells, i.e., at least 50% of CSR events require MMR, depending on the isotype (27, 30–34). To Downloaded from test this hypothesis, we analyzed S␮ DSBs in B cells from mice deficient in MMR proteins that have the WT S␮ region or the S␮TR deletion. Altogether, the data support the hypothesis that distal SSBs are converted by MMR into DSBs, constituting an important step in CSR. http://www.jimmunol.org/ Materials and Methods Mice AID-deficient mice were obtained from T. Honjo (Kyoto University, Kyoto, Japan). Msh2-deficient mice were obtained from T.