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

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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 © 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 , which are excised by 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 (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 , 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. Mak (Univer- sity of Toronto, Toronto, Canada). Mlh1- and Pms2-deficient mice were FIGURE 1. AID-dependent S␮ DSBs are detected in G -, but not in S/G / obtained from R. M. Liskay (Oregon Health Sciences University, Portland, 1 2 OR). XPA-deficient mice were obtained from H. van Steeg (National In- M-phase, cells. A, Splenic B cells were activated for 2 days, stained with stitute of Health, Bilthoven, The Netherlands) (35). These lines have all Hoechst 33342, and sorted into G1 and S/G2/M populations. A representative ϩ/Ϫ been extensively backcrossed to C57BL/6. xpa mice were bred to sorting profile is shown. B, LM-PCR was performed on DNA from viable by guest on September 27, 2021 ungϩ/Ϫ mice (obtained from D. Barnes and T. Lindahl, London Research sorted cells activated as indicated. Three-fold dilutions of 7200 cell equivalents Institute, London, U.K.) to generate doubly heterozygous mice, the breed- were amplified. PCR amplification of the GAPDH gene (except for the highest Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ ing of which generated wild-type (WT), xpa , ung , and xpa input) is shown below the blots as an internal control for template input. The Ϫ/Ϫ ␮ ϩ/Ϫ ung littermates used for the experiment shown in Fig. 3. S TR mice figure shown is representative of two experiments. were bred to mlh1ϩ/Ϫ and to msh2ϩ/Ϫ mice to generate double knockouts. WT (ϩ/ϩ) littermates from MMR-deficient mice were used as controls for all experiments (except that shown in Fig. 3) and the results were identical for all WT littermates. All mice were housed in the same room of the Western blotting Institutional Animal Care and Use Committee-approved specific pathogen- To prepare nuclear extracts, cells were resuspended in hypotonic buffer (10 free facility at University of Massachusetts Medical School and were bred mM HEPES (pH 8), 1 mM EDTA, 10 mM KCl, 0.1 mM EGTA, 1 mM and used under guidelines formulated by the University of Massachusetts DTT, 2 ␮M pepstatin, and complete protease inhibitor mixture); after a Animal Care and Use Committee. 15-min incubation on ice, cells were lysed by addition of Nonidet P-40 to a final concentration of 0.625%. After centrifugation at 12,000 rpm in a B cell isolation and culture microfuge for 10 min, the pelleted nuclei were washed once with hypotonic buffer and resuspended in hypertonic buffer (20 mM HEPES (pH 8), 1 mM Spleen cells were dispersed and RBCs lysed in Gey’s solution followed by EDTA, 400 mM NaCl, 1 mM EGTA, 1 mM DTT, 2 ␮M pepstatin, and T cell depletion with a mixture of anti-T cell Abs, as described previously complete protease inhibitor mixture). Protein content of nuclear extracts (30). Cells were cultured at 1 ϫ 105/ml in 6-well plates and activated to ␮ was determined using the Bradford assay (Bio-Rad). Proteins were elec- induce CSR. All cultures contained LPS (50 g/ml; Sigma-Aldrich) and trophoresed on 10% SDS-polyacrylamide gels or 4–20% gradient SDS- human BLyS (100 ng/ml), Human Genome Sciences. IL-4 (800 U/ml) was polyacrylamide gels (Bio-Rad), and blotted onto Immobilon-P polyvinyli- added to induce switching to IgG1; IFN-␥ (10 U/ml) for IgG2a; dextran ␮ ␦ dene fluoride membranes (Millipore). Immunoblotting was performed sulfate (30 g/ml; Amersham Biosciences) for IgG2b; anti- -dextran (0.3 using rabbit polyclonal Abs against AID and UNG (rabbit anti-peptide ng/ml) for IgG3, and to induce IgA switching, TGF-␤ (2 ng/ml), IL-4 (800 ␦ amino acids 280–295 from mouse UNG), APE1 (36), MSH2 (sc-494), U/ml), IL-5 (1.5 ng/ml; BD Biosciences), and anti- -dextran (0.3 ng/ml) MSH6 (sc-10798), MLH1(sc-582), and GAPDH (Santa Cruz Biotechnol- were added. Fixing of cells and staining of cell surface Abs were performed ogy), and monoclonal mouse anti-TBP1 (Abcam) followed by goat-anti- as described previously (30), except the results were analyzed by FlowJo rabbit or donkey anti-mouse-HRP (Santa Cruz Biotechnology) and ECL software. substrate (Pierce). Cell cycle sorting UNG assay Splenic B cells were cultured for 2 days as described above, after which Whole cell extracts were prepared from splenic B cells activated for 2 days, cell density was adjusted to 1 ϫ 106 cells/ml and stimulated B cells were washed in ice-cold PBS, and lysed in buffer I (10 mM Tris-HCl (pH 7.8), incubated for 90 min at 37°C with 3.5 ␮g/ml Hoechst 33342 (Invitrogen 200 mM KCl with Complete protease inhibitors (Roche)). An equal vol- Life Technologies) in HBSS containing 1% FCS. Cells were finally resus- ume of buffer II was added (buffer I with 2 mM EDTA, 40% glycerol, 0.2% pended in 1 ml of HBSS with 1% FCS; 7-aminoactinomycin D was added Nonidet P-40, and 2 mM DTT added) before tumbling for1hat4°C. (0.6 ␮g/ml) and cells were sorted by flow cytometry based on DNA content Supernatants were stored at Ϫ80°C. Nuclear extracts were made from using a UV laser-equipped FACSVantage SE (BD Biosciences). sorted cells by lysis in buffer A (10 mM HEPES (pH 8), 10 mM KCl, 0.1 6066 CELL CYCLE AND MISMATCH REPAIR-DEPENDENT S REGION DNA BREAKS

140

A 120

100

80

60

40

20

0 percent of WT switching IgG1 IgG2a IgG2b IgG3 IgA B 37.7 WT0.12 AID1.15 UNG

UNG 33.2 XPA 1.4 XPA IgG1 Downloaded from FIGURE 2. The proteins important for CSR are present in all phases of the cell cycle in activated B cells. A, Splenic B cells were activated for 2 IgM days, stained with Hoechst 33342, and sorted into G1, S, and G2/M pop- ulations. B–D, Western blots of extracts from sorted cells. B and C, A total of 35 ␮g of nuclear or cytoplasmic extract, as indicated, electrophoresed on C 17.8 WT0.06 AID0.39 UNG Ϫ/Ϫ Ϫ/Ϫ a gradient gel. ung and aid control extracts are from unsorted B http://www.jimmunol.org/ cells. D, Five micrograms of nuclear extract electrophoresed on a 10% polyacrylamide gel. E, UNG assay. Five-fold dilutions (10, 50, and 250 ng) of whole cell extracts from unsorted cells or nuclear extracts from cells 32 sorted as in A were added to a [ P]end-labeled uracil-containing oligonu- 11.7 XPA 0.37 UNG cleotide. Upper band, Uncleaved oligo; lower band, cleaved product. XPA IgG2a mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2 ␮M pepstatin, IgM and Complete protease inhibitor) with 0.625% Nonidet P-40. Nuclei were FIGURE 3. CSR is not reduced in XPA-deficient B cells. A and B, Cells by guest on September 27, 2021 pelleted and extracted with buffer C (20 mM HEPES (pH 8), 400 mM Ϫ Ϫ from xpa / mice were cultured for 4 days with LPS and cytokines to NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 40% glycerol, 0.5 ␮M pepstatin, and Complete protease inhibitor). Extracts were incubated with induce switching to the indicated isotypes and cells were analyzed by flow a[32P]5Ј end-labeled uracil-containing oligonucleotide (5Ј-GATTC cytometry for surface Ig. Data from four experiments were normalized to CCCATCTCCTCAGTTTCACT/ideoxyU/CTGCACCGCATG-3Ј; IDT) in the percent of switching by B cells from WT littermates (shown as 100%) Ϯ BER buffer (40 mM HEPES-KOH (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, and the average SEM is shown. B and C, Examples of flow cytometric ␤ 0.1 mM EDTA, 2 mM NaVO4, 50 mM NaF, 25 mM -glycerophosphate, analyses for CSR to IgG1 (B) and IgG2a (C). Switching to all four IgG 2 mM sodium pyrophosphate) at 37°C. After 1 h, NaOH was added to 0.1 isotypes and IgA were also analyzed with similar results. All mice used M and reactions were heated to 95°C for 7 min and electrophoresed on an were littermates with the exception of aidϪ/Ϫ. 8 M urea, 15% polyacrylamide bis (19:1) gel. Genomic DNA preparation and linker ligation-mediated PCR (LM-PCR) C57BL/6 12 (GenBank Accession number AC073553) with numbering starting at nt 136,645. This is the 5Ј S␮ primer-binding site and After culture for 2 days, viable cells were isolated by flotation on Ficoll/ ϳ800 nt upstream of the beginning of the TRs. Hypaque gradients (␳ ϭ 1.09), or lympholyte (Cedarlane Laboratories), cells were imbedded in low-melt agarose, and DNA was isolated and used for LM-PCR as described (15). Briefly, DNA was ligated overnight to Results linker, which was prepared by annealing 5 nM each of LMPCR.1 (5Ј- AID-dependent DSBs are predominantly detected in the G1 GCGGTGACCCGGGAGATCTGAATTC-3Ј) and LMPCR.2 (5Ј-GAAT phase of the cell cycle TCAGATC-3Ј)in300␮lof1ϫ ligase buffer, resulting in a double- stranded oligo with a 14-nt single-strand overhang that ligates A possible mechanism for conversion of SSBs into DSBs is DNA unidirectionally. Ligated DNA samples were assayed for GAPDH by PCR replication, and many of the proteins involved in CSR are associated to adjust DNA input before LM-PCR. The primer 5Ј S␮ (5Ј-GCA with DNA surveillance and repair during replication, e.g., MMR pro- GAAAATTTAGATAAAATGGATACCTCAGTGG-3Ј) was then used in teins and UNG (37). To determine when AID-dependent DSBs are conjunction with linker primer (LMPCR.1) to amplify DNA breaks. Three- ␮ fold dilutions of input DNA were amplified by HotStar Taq (Qiagen). PCR made, we examined the cell cycle regulation of S DSBs. DSBs products were run on 1.25% agarose gels and vacuum blotted (VacuGene dependent on AID and UNG are induced in the S regions of B cells XL; Pharmacia) onto nylon membranes (GeneScreen Plus; PerkinElmer). activated in vitro to undergo CSR and can be detected by linker LM- Blots were hybridized at 37°C overnight with an S␮ -specific oligonucle- PCR (15). We activated splenic B cells for 2 days with LPS and IL-4 ␮ Ј otide probe ( probe, 5 : AGGGACCCAGGCTAAGAAGGCAAT) end to induce IgG1 CSR or LPS and anti-␦-dextran (a-␦-dex) to induce labeled with [␥-32P]ATP and washed at 55°C with 2ϫ SSC/0.1% SDS. IgG3 CSR, stained with Hoechst 33342, and then sorted cells into G1

Cloning, identification, and sequence analysis of PCR products and S/G2/M fractions on the basis of DNA content (Fig. 1A). Dead LM-PCR products were cloned into the vector pCR4-TOPO (Invitrogen cells were excluded by 7-aminoactinomycin D staining. At 48 h, there Life Technologies) and sequenced by Macrogen using T3 and T7 primers. are no undivided cells in these cultures, as determined by CFSE stain- Cloned breaks in S␮ were aligned with germline S␮ sequenced from ing (our unpublished data). As shown in Fig. 1B, AID-dependent The Journal of Immunology 6067 Downloaded from http://www.jimmunol.org/

FIGURE 4. DSBs in S␮ are reduced in MMR-deficient splenic B cells. A, A representative Southern blot of S␮ LM-PCR products from B cells lacking the indicated protein, induced with LPS and IL-4. PCR amplification of the GAPDH gene is shown below the blots as an internal control for template input: 3-fold dilutions of 2400 cell equivalents. B, Quantification of DSBs in B cells lacking MMR or AID relative to DSBs in WT cells. Autoradiographs were scanned to measure total density in all three lanes for each sample, and results were normalized relative to density of WT bands in each experimental set. Error bars indicate SEM. The apparent discrepancy between the numbers by guest on September 27, 2021 and intensities of breaks in the pms2Ϫ/Ϫ and aidϪ/Ϫ samples between the blot shown in A and the compiled results in B is because in the other pms2Ϫ/Ϫ experiment there were more DSBs (data not shown). FIGURE 5. S␮ DSBs are greatly reduced in B cells lacking both S␮TRs and Msh2. S␮ LM-PCR products (A and B) amplified as in Fig. 4 from cells with the indicated deficiencies activated as indicated. C, Quantifica- DSBs are detected almost exclusively in the G1 fraction in cells ac- tion of DSBs by densitometry (as in Fig. 4B) of the indicated number of tivated under either condition. There are a few breaks detected in experiments. Error bars represent SEM. The aidϪ/Ϫ histogram presents the

S/G2/M phase, but there are about as many in AID-deficient B cells. same data shown in Fig. 4B. These data are therefore inconsistent with the hypothesis that DSBs de- tected in S␮ in switching B cells are due to replication across SSBs. important for CSR are present in all phases of the cell cycle, and Ϫ/Ϫ Although a few DSBs are detected in aid cells, they do not specifi- specifically in G1 phase when AID-dependent DSBs are detectable. cally occur at the G:C in the AID target hot spots, unlike DSBs in WT cells (15). They might be due to mechanically induced breaks or G1-phase cells have UNG activity

apoptosis occurring during the procedure, or perhaps due to replication. UNG is not present in resting (G0) cells (14) and has been proposed to act in S phase at replication forks during CSR and somatic hyper- Proteins important for CSR are expressed in G1-phase cells mutation (40). G1-phase B cells have not been examined for UNG

We then asked whether the levels of AID, UNG, APE1, and MMR activity. We prepared nuclear extracts from G1- and S-phase B cells proteins are regulated by the cell cycle. B cells activated with LPS and activated for 2 days and assayed UNG activity by an oligonucleotide ␦ a- -dex were sorted into G1,S,andG2/M fractions (Fig. 2A) and cleavage assay (Fig. 2E). Activity is clearly detectable in as little as 10 extracts were prepared for Western blotting (Fig. 2, B–D). AID pro- ng of nuclear extract from G1-phase cells, which has as much activity tein is difficult to detect in nuclear extracts, but is expressed in the as extracts from S-phase cells. As this substrate is single stranded, the cytosol in all phases of the cell cycle (Fig. 2C). UNG is abundant both predominant activity detected here is likely to be due to UNG and not

in the nucleus (Fig. 2B) and cytoplasm (Fig. 2C)ofG1- and S-phase SMUG-1, as SMUG-1 has 800-fold lower activity on ssDNA than cells. The multiple UNG bands observed in nuclear extracts represent UNG (kcat/Km) (41). From these combined results, we conclude that ␮ ␮ differently phosphorylated species (38). The Ab should also detect the AID-dependent DSBs in S are made and resolved in S in G1 phase mitochondrial form of UNG in the cytosol. We observed a decrease in and are not due to replication across SSBs. UNG in the cytosol as the cells progress through cell cycle, consistent with a report that UNG is down-regulated in late S phase (39). APE1 The role of Ercc1 in CSR does not involve the NER pathway and the MMR proteins Msh2, Msh6, and Mlh1 are all expressed in the We next asked whether NER might contribute to the generation of nucleus throughout the cell cycle (Fig. 2D). We conclude that proteins DSBs during CSR. We have previously shown a minor role for 6068 CELL CYCLE AND MISMATCH REPAIR-DEPENDENT S REGION DNA BREAKS Downloaded from http://www.jimmunol.org/

FIGURE 6. S␮ (left) and S␮TRϪ/Ϫ (right) sequences. The section in S␮ shown in black (and red) is deleted from the S␮TRϪ/Ϫ intron. The blue (and by guest on September 27, 2021 red) section is present in both S␮ and S␮TRϪ/Ϫ sequences. The black segment in the S␮TRϪ/Ϫ intron represents the loxP insertion. All GYW/WRC AID hot spots are underlined and GCT motifs are shown in red. Arrows indicate the sites of DSBs from cloned LM-PCR products. Bracketed nucleotides roughly indicate the TR region of S␮.

Ercc1 in CSR (20). The heterodimer Ercc1-XPF is an essential CSR. A representative Southern blot of LM-PCR fragments am- component of the NER pathway, but Ercc1-XPF can also act as a plified with a 5Ј S␮-specific primer from WT, msh2Ϫ/Ϫ, mlh1Ϫ/Ϫ, structure-specific endonuclease in the absence of the rest of the NER and pms2Ϫ/Ϫ B cells is shown in Fig. 4A. Blunt DSBs in MMR- pathway, cutting at the junction of ssDNA and dsDNA (19). Here, we deficient cells are reduced compared with WT cells analyzed in analyzed CSR in vitro in B cells from mice lacking XPA, a protein parallel. Quantitation by densitometry scanning of the autoradio- essential for NER. We found no reduction in CSR in xpaϪ/Ϫ B cells graphs from several experiments demonstrates that MMR defi- relative to cells from WT littermates (Fig. 3A). In the absence of ciency results in ϳ20–50% of the number of S␮ DSBs in WT cells UNG, it seemed possible that the accumulation of uracils might result (Fig. 4B). Thus, blunt DSBs induced in S␮ during CSR are re- in distortion of the DNA helix sufficient to create a substrate for NER, duced in MMR-deficient cells to a degree consistent with the re- so we also analyzed CSR in ungϪ/ϪxpaϪ/Ϫ B cells CSR is severely duction observed in CSR. reduced in B cells deficient in UNG, as reported by others (13, 14), As described in the introduction, CSR in B cells lacking the Ig but still detectable relative to AID-deficient B cells. However, CSR is S␮ TR region (S␮TRϪ/Ϫ) is severely reduced (90–95%) in the not further reduced in ungϪ/ϪxpaϪ/Ϫ B cells relative to B cells from absence of Msh2 or Mlh1. We used LM-PCR to determine whether ungϪ/Ϫ littermates (Fig. 3, B and C). In this experiment, there was a this might be due to a decrease in DSBs. As shown in Fig. 5, DSBs reduction in IgG1 and IgG2a CSR in xpaϪ/Ϫ cells, but this was not in S␮TRϪ/Ϫ B cells are detected at only a slightly lower frequency typical as can be seen from the compiled data in Fig. 3A. We conclude than in WT cells (65% of WT), consistent with the 2-fold reduction that the minor role played by Ercc1-XPF in CSR most likely involves in CSR in these B cells (24), but when the cells also lack Msh2 or its ability to cut at the junction of ssDNA and dsDNA (discussed Mlh1, DSBs are reduced to only 20% of that seen in WT cells. The below and in Ref. 29), and does not involve other components of the number of DSBs detected in Msh2/S␮TR and Mlh1/S␮TR double NER pathway. knockout cells is almost as low as in AID-deficient cells.

S-region DSBs are reduced in MMR-deficient B cells Sites of DSBs in S␮TR-deficient cells The hypothesis that SSBs are converted to DSBs in S regions by Relative to the WT S␮ region, the S␮TRϪ/Ϫ intron has a 2-fold MMR predicts fewer DSBs in S␮ in MMR-deficient B cells rel- lower incidence of AID hot spots (WRC/GYW, where the under- ative to WT cells. To test this prediction, LM-PCR was performed lined nucleotide is the target site), and the strongly preferred hot- on genomic DNA isolated from B cells 2 days after induction of spot GCT (11, 33) is 3.6-fold less frequent than in WT S␮ (Fig. 6). The Journal of Immunology 6069

Table I. DSBs occur preferentially at AID hot spots in WT and (44), and that a specific protein complex binds near the transcrip- S␮TRϪ/Ϫ B cells ␥ tional initiation site for germline 1 transcription during G1/early S phase, but not during G2/M phase in splenic B cells activated to WT S␮ TRϪ/Ϫ undergo CSR (45). We also found that AID, UNG, and MMR Frequencya Frequencya proteins are present in the G1 phase of the cell cycle, and that extracts from G1-phase B cells possess UNG activity. G:C 83.5%b,c 54.9% 83.9%b,c 47.4% MMR proteins could convert SSBs into DSBs in the course of A:T 16.5% 45.1% 16.1% 52.6% normal MMR activity if nicks are present on both strands. Both GYWe 40.5%c 12.4% 51.6%c 6.4% GCT 35.4%c 10.6% 29.0%c 2.9% AID and UNG prefer ssDNA substrates and appear to act on both GCA 2.5% 0.4% 3.2% 0.8% DNA strands during transcription (1, 12, 22, 46). We hypothesize GTT 1.3% 0.7% 9.7% 1.8% that the DNA duplex reforms before all of the dUs can be removed GTA 1.3% 0.7% 9.7% 0.9% by UNG, and MMR would then compete more effectively than Total 79 breaks nt 1–2000d 31 breaks nt 1–1439d UNG for repair of dUs in duplex DNA. AID is a highly processive a The frequency at which the nucleotide or sequence motif occurs in the sequence enzyme and although UNG has a very high catalytic rate, it ap- analyzed (S␮ or S␮TRϪ/Ϫ). b Percent of DSBs located at the indicated base pair or sequence motif. pears to be unable to remove all the dUs introduced by AID into c Significantly targeted relative to the DNA sequence, p Յ 0.012 (Fisher’s exact S regions (6, 12, 22, 23, 41). UNG is likely, however, to create test). d numerous abasic sites, which could then be nicked by APE to nt 1, Nucleotide 136,645 in the chromosome 12 sequence from C57BL/6 Ј Ј (GenBank Accession AC073553). generate entry sites for excision by Exo1 in the 5 to 3 direction. e The underlined G, G:C bp at which the DSB occurs within the hot spots, reading If excision continued until a SSB on the opposite strand is reached, Downloaded from the top strand sequence. a DSB would be formed. A similar model has been proposed for Escherichia coli dam cells exposed to the methylating agent MNNG, in which DSBs arise when MMR excision on one strand Ϫ Ϫ We asked whether the DSBs in S␮TR / cells occur preferentially encounters a nick generated by APE on the other (47). Depending at these remaining hot spots by cloning and sequencing LM-PCR on the orientation of the SSBs relative to each other, some DSBs ␮ Ϫ/Ϫ products from WT and S TR B cells. Table I presents the created by MMR activity will be blunt and some will have 5Ј or 3Ј http://www.jimmunol.org/ percent of DSBs occurring at the G:C or A:T base pair, at all AID overhangs. 5Ј overhangs can be filled in by polymerase to become hot spots (GYW), and at the four different GYW motifs (GCT, blunt, and we have proposed that Ercc1-XPF can remove 3Ј over- GCA, GTT, and GTA), as well as the frequency of occurrence of hangs (20, 29). Ercc1-XPF cuts 3Ј overhangs at the junction of Ϫ Ϫ these motifs in the S␮-region sequence in WT and S␮TR / cells. ssDNA and dsDNA without requiring additional NER proteins The percentage of DSBs located at GYW sequences is increased in (48). Consistent with Ercc1-XPF acting independently of NER in Ϫ Ϫ S␮TR / B cells (51.6% compared with 40.5% in WT), support- CSR, we found that XPA, an essential NER protein, is not in- ing the importance of this sequence for AID targeting. volved in CSR even in the absence of UNG, where helix-distorting Ϫ Ϫ Further analysis of the breakpoints used in the S␮TR / intron lesions recognizable by NER might form. support previous conclusions that GCT/AGC is the strongest AID If the SSBs are the results of AID-instigated lesions, the result- by guest on September 27, 2021 hot spot. The preference for breaks at GCT in WT S␮ is 3.3-fold ing blunt DSBs would occur preferentially at the G:C base pair in over random (35.4% of DSBs at GCT vs 10.6% frequency of GCT AID hot spots, which is consistent with the location of DSBs in S␮ in the sequence, p Ͻ 0.001). However, GCT occurs in the WT S␮ determined by LM-PCR (Table I) (15, 29). No significant differ- sequence at a much higher frequency than the other GYW hot ences were observed between the sites of S␮ DSBs in WT and Ϫ Ϫ spots (Table I). In the S␮TR / intron, the frequency at which MMR-deficient B cells (Table II). In the absence of MMR, we GCT occurs relative to the other GYW motifs is lower; interest- predict that the only DSBs formed would do so spontaneously ingly, DSBs at GCT occur 10-fold more often than predicted by from closely spaced nicks. The staggered DSBs produced in the the sequence (29% of DSBs are at GCT compared with 2.9% fre- absence of MMR could then be end processed as described above quency in the sequence, p Ͻ 0.001). These results confirm that the to form the blunt DSBs at the G:C base pair in AID hot spots preference of AID for GCT hot spots previously found in vitro detected by LM-PCR. The staggered ends can be detected by LM- with purified AID and from analyses of somatic hypermutation (6, PCR by pretreatment with T4 DNA polymerase before ligation 11, 12, 42, 43) is also true during CSR, and is even more apparent (15, 17). We find a 3-fold increase in DSBs after T4 treatment of when these motifs are less abundant. We hypothesize that the re- DNA from both WT and MMR-deficient cells (data not shown). Ϫ Ϫ duction in AID hot spots in S␮TR / B cells results in SSBs that It has been reported that 94% of S␮-S␥3 junctions in Msh2- are farther apart, and thus less likely to be sufficiently close to form deficient cells occur within the S␮ TRs, which contain numerous a DSB spontaneously. DSB formation and CSR are therefore more closely spaced GAGCT sequences, and only 5% occur 5Ј to the dependent on MMR in these mice. TRs, whereas in WT cells a greater proportion (17%) of the junc- tions occur 5Ј to the repeats (26, 27). These results predict that Discussion blunt DSBs in MMR-deficient B cells would localize preferentially The results from our studies support the hypothesis that distal to the S␮ TRs. However, we found a similar fraction of DSBs SSBs are converted into DSBs by MMR recognition and repair of occur 5Ј to the S␮ TRs in WT and msh2Ϫ/Ϫ cells, 42 and 40%, dU:dG mismatches introduced by AID. We obtained data arguing respectively. This difference from the previous reports might be against an alternative model for conversion of SSBs to DSBs, i.e., due to differences in the analysis methods. We used a PCR primer Ј ␮ replication across a SSB, as we detected very few DSBs in S/G2/M located at the 5 side of S , and extension would terminate at the ␮ phase cells. Instead, the AID-dependent DSBs occur in the G1 first break, favoring detection of breaks located upstream of S . phase. These results are in agreement with previous reports show- We consistently find fewer blunt DSBs in B cells from mice ing that AID-dependent ␥H2AX/Nbs1 foci colocalize with IgH lacking Mlh1 or Pms2 than in those lacking Msh2. This might be

loci during the G1/early S phase in splenic B cells activated to explained by the recent finding that Mlh1 decreases Exo1 proces- switch (18). They are also consistent with the finding that switch sivity (49). If Exo1 excises farther in the absence of Mlh1-Pms2, recombination is not accompanied by sister-chromatid exchange perhaps past the nick on the opposite strand, long single-strand 6070 CELL CYCLE AND MISMATCH REPAIR-DEPENDENT S REGION DNA BREAKS

Table II. Sites of DSBs in S␮ do not differ between WT and 3. Petersen-Mahrt, S. K., R. S. Harris, and M. S. Neuberger. 2002. AID mutates E. MMR-deficient B cells during CSRa coli suggesting a DNA deamination mechanism for antibody diversification. Na- ture 418: 99–104. 4. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F. W. Alt. 2003. Ϫ Ϫ Ϫ Ϫ WTb msh2 / / Frequencyc Transcription-targeted DNA deamination by the AID antibody diversification en- zyme. Nature 422: 726–730. G:C 83.5%d 83.3%d 93.8%d 54.9% 5. Dickerson, S. K., E. Market, E. Besmer, and F. N. Papavasiliou. 2003. AID A:T 16.5d 16.7% 6.2% 45.1% mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197: GYWe 40.5%d 43.3%d 43.8% 12.4% 1291–1296. GCT 36.7%d 36.7%d 25.0% 10.6% 6. Pham, P., R. Bransteitter, J. Petruska, and M. F. Goodman. 2003. Processive GCA 2.5% 3.3% 12.5% 0.4% AID-catalysed cytosine deamination on single-stranded DNA simulates somatic GTT 1.3% 0.0% 6.3% 0.7% hypermutation. Nature 424: 103–107. 7. Bransteitter, R., P. Pham, M. D. Scharff, and M. F. Goodman. 2003. Activation- GTA 1.3% 3.3% 0.0% 0.7% f g induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA Total 79 30 16 nt 1–2000 but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100: 4102–4107. a The data are reported as a percent of the DSBs at the indicated base pair or 8. Ramiro, A. R., P. Stavropoulos, M. Jankovic, and M. C. Nussenzweig. 2003. sequence motif. Transcription enhances AID-mediated cytidine deamination by exposing single- b WT (ϩ/ϩ) littermates from several DNA repair mutants, all of which gave stranded DNA on the nontemplate strand. Nat. Immunol. 4: 452–456. identical results. 9. Yu, K., F. Chedin, C. L. Hsieh, T. E. Wilson, and M. R. Lieber. 2003. R-loops c The frequency at which the nucleotide or sequence motif occurs in the S␮ at immunoglobulin class switch regions in the of stimulated B sequence. cells. Nat. Immunol. 4: 442–451. d Significantly targeted relative to the DNA sequence, p Յ 0.037 (Fisher’s exact 10. Stavnezer, J. 1996. Immunoglobulin class switching. Curr. Opin. Immunol. 8: t test). 199–205. e The underlined G indicates the G:C base pair at which the DSB occurs within the 11. Yu, K., F. T. Huang, and M. R. Lieber. 2004. DNA substrate length and sur-

hot spots, reading the top strand sequence. rounding sequence affect the activation-induced deaminase activity at cytidine. Downloaded from f Total number of DSB sites analyzed. J. Biol. Chem. 279: 6496–6500. g nt 1, Nucleotide 136,645 in the chromosomal 12 sequence from C57BL/6 12. Bransteitter, R., P. Pham, P. Calabrese, and M. F. Goodman. 2004. Biochemical (GenBank Accession AC073553). analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase. J. Biol. Chem. 279: 51612–51621. 13. Rada, C., G. T. Williams, H. Nilsen, D. E. Barnes, T. Lindahl, and M. S. Neuberger. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12: 1748–1755. tails and fewer blunt DSBs would be generated. Long single-strand 14. Imai, K., G. Slupphaug, W. I. Lee, P. Revy, S. Nonoyama, N. Catalan, L. Yel, http://www.jimmunol.org/ overhangs might also explain the increase in microhomology M. Forveille, B. Kavli, H. E. Krokan, et al. 2003. Human uracil-DNA glycosylase Ϫ/Ϫ Ϫ/Ϫ deficiency associated with profoundly impaired immunoglobulin class-switch re- found at CSR junctions from mlh1 and pms2 mice. In ad- combination. Nat. Immunol. 4: 1023–1028. dition, it has recently been shown that the Mlh1-Pms2 heterodimer 15. Schrader, C. E., E. K. Linehan, S. N. Mochegova, R. T. Woodland, and has endonuclease activity that introduces nicks on either side of the J. Stavnezer. 2005. Inducible DNA breaks in Ig S regions are dependent upon AID and UNG. J. Exp. Med. 202: 561–568. mismatch (50). This activity appears to be restricted to the previ- 16. Catalan, N., F. Selz, K. Imai, P. Revy, A. Fischer, and A. Durandy. 2003. The ously nicked strand (50), and so by itself would not result in DSBs block in immunoglobulin class switch recombination caused by activation-in- duced cytidine deaminase deficiency occurs prior to the generation of DNA dou- during CSR. Furthermore, as the Mlh1-Pms2 endonuclease does ble strand breaks in switch mu region. J. Immunol. 171: 2504–2509. not appear to be restricted to a specific DNA sequence, and S␮ 17. Rush, J. S., S. D. Fugmann, and D. G. Schatz. 2004. Staggered AID-dependent ␮ DSBs strongly favor the G:C base pair, this activity is unlikely to DNA double strand breaks are the predominant DNA lesions targeted to S in by guest on September 27, 2021 Ig class switch recombination. Int. Immunol. 16: 549–557. contribute significantly to S-region breaks. 18. Petersen, S., R. Casellas, B. Reina-San-Martin, H. T. Chen, M. J. Difilippantonio, Our results are consistent with the model that MMR is more P. C. Wilson, L. Hanitsch, A. Celeste, M. Muramatsu, D. R. Pilch, et al. 2001. important for CSR in situations when SSBs are limiting, as when AID is required to initiate Nbs1/␥-H2AX focus formation and mutations at sites ␮ of class switching. Nature 414: 660–665. AID targets are scarce. 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