Ig gene somatic hypermutation in mice defective for DNA polymerase {delta} proofreading

Angelika Longacre1, Tianhe Sun1, Robert E. Goldsby2, Bradley D. Preston3 and Ursula Storb1

1 Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA 2 Present address: Department of Pediatrics, University of California at San Francisco, San Francisco, CA 94143, USA 3 Present address: Department of Pathology, University of Washington, Seattle, WA 98195, USA

Correspondence to: U. Storb; E-mail stor{at}midway.uchicago.edu
Transmitting editor: E. A. Clark


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study is an investigation of the possible role of DNA polymerase (pol) {delta} with an inactivated exonuclease (exo) in somatic hypermutation (SHM). Analysis of endogenous heavy chain transcripts revealed no difference in mutation frequency and pattern between exo–/–, exo+/– and exo+/+ mice. The lack of an effect of the pol {delta} exo mutation on SHM could be due to: (i) normally pol {delta} is used in SHM, but the exo is prevented from proofreading, (ii) normally pol {delta} is used, but the decrease in fidelity of the exo pol does not increase hypermutation frequency enough to be detected, and (iii) pol {delta} is not used in SHM. Based on the finding in the exo–/– mice and the current understanding of the process of SHM, it is concluded that pol {delta} is not normally involved in creating the mutations. The majority of the mutated sequences obtained in this study, including many from the exo–/– mice, were from genes which had switched to a {gamma} heavy chain class. Thus, the pol {delta} proofreading activity is not required for class switch recombination (CSR). Genealogical trees observed with multiple mutated sequences of various Ig classes show that CSR and SHM occur intermingled during expansion of a cell clone, raising the possibility that they may occur at the same time.

Keywords: class switch recombination, polymerase {delta} exonuclease, somatic hypermutation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mechanism of somatic hypermutation (SHM) of Ig genes remains a mystery, but most models suggest the involvement of DNA polymerase(s) (pol) in at least one step of the process [reviewed in (1)]. In a model we have proposed, a mutator factor (MuF) loads onto an initiating RNA pol and is carried into the V region where it is deposited and creates a lesion in the DNA (2). The postulated MuF possibly is the recently discovered activation-induced deaminase (AID) (3,4). The subsequent steps would involve repair in which a single nucleotide or a short sequence of one DNA strand is replaced by a new sequence synthesized by a DNA pol which introduces occasional mutations. One way to test the various possibilities of error introduction is to alter the fidelity of a pol and then test its effect on SHM.

Mice homozygous for a pol {delta} (pol {delta}) exonuclease (exo) inactivating point mutation were generated by Goldsby et al. (5). In order to determine if pol {delta} is used during the postulated repair phase of hypermutation, we have analyzed endogenous IgH genes in these mice for differences in mutation frequencies or patterns.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Polymerase {delta} exo mice
Mice carrying a pol {delta} exo inactivating point mutation (amino acid D407A in the Exo II motif) (exo) were described previously (5). Chimeric mice carrying the mutant pol {delta} allele were generated by fusing recombinant SvJ-129 ES cells with C57BL/6J morulas. Subsequent crossing of chimeric males with C57BL/6J females generated heterozygous F1 mice whose interbreeding resulted in F2 homozygous (exo–/–), heterozygous (exo+/–) and wild-type (exo+/+) mice all with mixed SvJ-129/C57BL/6J genetic backgrounds. Two sets of three littermates with the three genotypes were analyzed by multiple RT-PCR assays.

Immunization of pol {delta} mice, spleen harvest and RNA isolation
Mice were immunized by i.p. injection of 2 x 108 sheep red blood cells in 100 µl PBS, with re-immunization on day 21. Spleens were harvested on day 24. Serum hemagglutination tests done at the time of harvest indicated that all the mice displayed an Ig response against sheep red blood cells. Spleen cells were released by pushing diced spleens through 70-µm nylon cell strainers (Fisher Scientific, Hanover Park, IL). Red blood cells were lysed by 30-s incubation in a hypotonic solution of 1 part RPMI:9 parts water, followed by a wash in 20–30 ml RPMI.

RNA was prepared from total spleen white blood cells using either RNA-Stat 60 (Tel-Test, Friendswood, TX) or the PolyAPure Kit (Ambion, Austin, TX), both of which yielded high quality RT-PCR products.

Flow cytometry
Some of the spleen white blood cells were analyzed by flow cytometry for the presence of lymphocyte-specific surface markers. The antibodies used were as follows: CD19–FITC, CD19–biotin followed by streptavidin–CyChrome, B220–biotin followed by streptavidin–CyChrome, CD43–phycoerythrin (PE), CD25–PE, IgM–PE, IgDa– and IgDb–FITC (both allotypes), CD3–PE, GL7–FITC (all from PharMingen, San Diego, CA); peanut agglutinin (PNA)–FITC (Sigma, St Louis, MO); PNA–biotin (Vector Laboratories, Burlingame, CA) followed by streptavidin–PE (PharMingen). Flow cytometry was performed on a Becton Dickinson (San Jose, CA) FACScan in the Immunology Applications Core Facility at the University of Chicago and analyzed with CellQuest software.

Selective amplification of VH-S107 family transcripts from mouse spleen.
cDNA synthesis and amplification of the VH genes were carried out as described previously (6) using the T15VH1 primer (5'-TGTGAGGTGAAGCTGGTGGAATCTG-3') and a Cµ primer MCH1 (5'-CTCGCAGGAGACGAGGGGGA-3) or C{gamma} primer GXCH1 (5'-CCAGGGGCCAGTGGATAGAC-3') which primes all {gamma} genes. RT-PCR products were purified using a Qiagen (Valencia, CA) Nucleotide Removal Kit, resuspended in 5 µl elution buffer and the entire reaction cloned into the pCR-Script vector using the Stratagene (La Jolla, CA) PCR-Script Cloning Kit. Individual colonies were screened by PCR with internal primer 5'107int (5'-AACAGAGTACAGTGCA TCTG-3') and the GXCH1 primer. Colonies containing the VH S107 family genes yielded a PCR product of ~250 bp. This screen eliminates cDNA clones that are not derived from the VHS107 family (7).

Clones that passed the S107 screen were sequenced by the University of Chicago Cancer Research Center DNA Sequencing Facility using an ABI (Applied Biosystems, Foster City, CA) Prism automated DNA sequencer. Sequence analysis was performed with Sequencher software.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B cell development in pol {delta} exo–/– mice
The FACS profiles of B lymphocytes and their precursors in the bone marrow of pol {delta} exo–/– were indistinguishable from those of pol {delta} wild-type mice (not shown). Also, in the spleen the overall pattern of T and B cells was normal.

Somatic mutation in immunized spleen B cells
Total spleen mRNA was tested for the presence of mutated Ig genes by using PCR primers specific for V heavy chain genes of the S107 family, and for µ and various {gamma} constant regions. In all three pol {delta} exo genotypes (+/+, +/– and –/–) the µ mRNAs had relatively low levels of somatic mutations in the V region (Table 1, Fig. 1 and supplementary data available at International Immunology Online). In the +/+ mice, five of 10 µ sequences (50%) had no mutations, the other five had only one to five mutations. In the +/– mice, 13 of 16 sequences (81%) had no mutations, the other three had one to three mutations. In the –/– mice, 15 of 18 sequences (83%) had no mutations, the other three had one mutation each. The mutation frequencies in the µ genes (0.33, 0.14 and 0.06 x 10–2, Table 1) are within the normal range of mutations in S107 V genes associated with the Cµ constant region (7). No mutations were found in the short segment nucleotides of Cµ region sequenced. The mutation indices (MI) (8) of the V region mutations were neutral to moderately hot. Thus, SHM of heavy chain µ genes in pol {delta} exo–/– mice is normal.


View this table:
[in this window]
[in a new window]
 
Table 1. Summary of somatic mutations
 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. Model of SHM. E, Ig enhancer; IEBP, Ig enhancer binding protein(s); RNAP, RNA pol II; X, point mutation.

 
In the IgG mRNAs of all three pol {delta} genotypes, robust numbers of somatic point mutations were observed in V-S107 sequences (supplementary data available form the authors). Every one of the eight mRNAs from +/+ mice had mutations, with a range of four to 11 per 275 nucleotides sequenced. In +/– mice, two of 22 {gamma} mRNAs showed no mutations, the rest had one to 11 point mutations in 275 nucleotides sequenced. In the –/– mice, one of 14 {gamma} mRNAs had no mutation, the rest had two to 12 mutations per 275 nucleotides. The mutation frequencies in the three genotypes were 2.45, 1.70 and 1.87 x 10–2 respectively (Table 1). These frequencies are slightly higher than previously observed (6,7). Only a total of 294 nucleotides of C{gamma} regions were sequenced from the pol {delta}–/– mice, but no mutations were found. Given the mutation frequency in the V region sequences, five to six mutations would be expected in the C region if V and C were equally susceptible in these mice. The lack of C region mutations suggests that the mutations in the V region were due to SHM, not to the pol {delta} exo defect. Clearly, the pol {delta} exo–/– mice are not altered in SHM. The mutation indices of the mutated nucleotides in the VH region of Ig genes for all three pol {delta} genotypes are in the lower hotspot range (Table 1, V average MI 1.31, 1.44 and 1.35). This is essentially the same as the MI observed in the human BCL-6 genes from three different donors (1.34, 1.31 and 1.39) and in IgG genes of one donor (1.31) (9).

In the {gamma} sequences, as expected, in the wild-type and exo+/– mice the ratio of replacement to silent mutations (R/S) is higher in the complementarity-determining regions (CDR) than in the framework regions (FWR) (Table 1). This suggests that the B cells carrying these mutations were selected for functional changes that presumably increase antibody affinity. In the pol {delta} exo–/– mice, on the other hand, the R/S is low in both the CDR and the FWR (see Discussion).

Somatic mutation and class switch recombination (CSR) may occur at the same time
Many of the mutated sequences appear to be clonally related and can be grouped into sequence pedigrees (supplementary data available online at International Immunology Online). The cells within each pedigree are of the same cell clone since they have identical VDJ joint nucleotides and share mutations. Two pedigrees were obtained from pol {delta} exo–/– mice; thus, the SHM process continues over several cell generations also in these exo deficient mice. Multiple pedigrees in all three pol {delta} genotypes suggest that SHM and CSR are intermingled processes (see Discussion).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pol {delta} and SHM
The analysis presented here was based on the possibility that pol {delta} may be involved in creating errors during the DNA repair phase of SHM. Pol {delta} is one of the two pols required for cell cycle DNA replication (the other is pol {epsilon}). Pol {delta} has also been shown to contribute to all the major avenues of DNA repair [nucleotide excision repair, base excision repair, mismatch repair and recombination, including V(D)J recombination; reviewed in (10)].

Inactivation of the 3'–5' exo by one of several point mutations in the exo domains of pol {delta} inhibits its proofreading capacity, while retaining the pol activity (1113). Mice with the homozygous knock-in of a pol {delta} exo mutation produce normal levels of pol {delta} and are fertile; however, they develop tumors early in life and 70% of the mice are dead by 11 months of age (5).

We considered that if pol {delta} is involved in SHM, the lack of its exo activity may increase the frequency of mutations in SHM or alter its pattern. Of course, the mutant pol {delta} will be more error prone when it is involved in DNA replication. However, this increase from about one error in 109 nucleotides to one in 106 nucleotides during the S phase of the cell cycle will not be detected in V genes of B cells which divide perhaps 20 times during SHM (14). This would only result in one point mutation in ~200 sequences.

The findings in this study clearly show that the impaired proofreading capacity of pol {delta} does not result in an increase in the rate of SHM nor does it affect the pattern of mutation. In IgG genes, the frequency of mutations is in the same range as in pol {delta} wild-type mice and the MI of the positions targeted for mutation is also the same. There is some decrease in –/– mice in the ratio of replacement to silent mutations (R/S) in both the CDR and the FWR. This may be due to the high proliferative activity of the mutating cells which may accumulate deleterious mutations genome-wide in each cell cycle in the pol {delta} exo–/– mice. Perhaps, B cells with replacement mutations that are favorable to antigen binding are stimulated to continue to proliferate and therefore have a greater chance to die because of genome-wide errors.

The absence of an influence on SHM by the proofreading defect may be explained in several ways which we will consider based on our model of SHM (2) (Fig. 1). We assume in this model that a MuF initiates SHM by loading onto the transcription complex at an Ig V gene promoter, traveling with the RNA pol during transcript elongation and being deposited on the DNA within 1–2 kb from the promoter. A likely candidate for MuF is AID which appears to be a DNA cytidine deaminase (1517). AID would potentially be able to deaminate any cytosine in the DNA near its deposition site. The resulting uracil would either be excised by a uracil glycosylase or copied into A during the next S phase, resulting in a C/G to T/A transition in one daughter cell. The replicating DNA pol in this step is likely pol {delta} which would faithfully insert an A across the U. The fact that in ung–/– mice most mutations from C/G are transitions to T/A suggests that they do occur during normal cell cycle replication. Inactivation of the pol {delta} exo would not cause a high enough mutation frequency to be detected (see above). An abasic site created by a uracil glycosylase could either be repaired by base excision repair or induce an error-prone bypass pol during the next S phase (18).

In these scenarios, pol {delta} could be one of the DNA pols involved in repairing the abasic site created by a DNA glycosylase. So far, none of the following DNA pol, ß, {eta}, µ or {lambda}, have been found to be essential for SHM (1921). All or some of these appear, nevertheless, to be involved and, in fact, the deficiency in pol {eta} has been shown to lead to an altered mutation spectrum (21). Inhibition of pol {zeta} decreases SHM levels (22,23). Pol {iota} appears to be required for high levels of SHM in a human B cell line (24). Given the two major patterns of hotspots, RGYW/WRCY and WA/TW, it is likely that at least two different pols are involved in SHM (8,25). The lack of an effect of the pol {delta} exo mutation on SHM could be due to the following: (i) normally pol {delta} is involved, but the decrease in fidelity of the exo pol does not increase hypermutation frequency enough to be detected by our assay, (ii) normally pol {delta} is involved in SHM, but the exo is prevented from proofreading, and (iii) pol {delta} is not creating somatic mutation.

The possibility that pol {delta} is normally involved in SHM and uses its exo activity independent of cell cycle DNA replication (e.g. during base excision repair) is unlikely, given our findings. The exo within pol {delta} increases its fidelity 130- to 1900-fold (13). It would be expected that with exo pol {delta} such an increase in primary mutations would be detected, assuming that the SHM process occurs many times in each cell cycle.

It appears unlikely that the pol {delta} exo would normally be prevented from proofreading during SHM. The exo consists of three domains (exo I, II and III) which are embedded in the catalytic region of the pol. The exo domains of pol {delta} could have been targets if AID was an RNA editing cytidine deaminase, as originally suggested (3,4). However, it appears now that AID is a DNA deaminase which targets Ig genes directly (16). It is highly unlikely that AID would also target the pol {delta} gene since it is required for high-fidelity replication of the genome and most B cells that have undergone SHM become long-lived memory cells.

Thus, there is no indication that pol {delta} creates mutations in SHM (except when faithfully copying uracil that arose by AID activity). As suggested in the model shown in Fig. 1 and reviewed elsewhere (17), the repair phase of SHM is likely to involve naturally error-prone DNA pols. It is not understood how pol {delta} would be excluded in this step.

SHM and CSR
The relationship between SHM and CSR has been investigated by others (2628). Two studies concluded that SHM precedes CSR (2729). In fact, one of these studies (28) postulated that CSR terminates SHM. We have observed new mutations that possibly occurred before as well as after CSR in the same cell lineage, suggesting that the two processes may be intermingling. The data support the stochastic model of Weigert’s laboratory (29). It is also interesting to note that in most of the pedigrees the {gamma} isotype does not change, suggesting that conditions which favor SHM do not necessarily induce further CSR.

It is not known from our and other data whether a given cell can undergo both processes. The question is whether during CSR there is always a chance for mutating the V region [besides the region around the switch sites (30)] and vice versa. Clearly, the cell uses different signals to access different CH genes. Thus, switching, even in the presence of AID, should not be activated unless the proper cytokines are produced. If only the VDJ–Cµ region is activated, the cell should undergo SHM without CSR in the presence of AID. However, once a downstream switch region is activated, the cell should be able to undergo both switching and SHM. Since activation of switch region transcripts can occur without AID expression (3) it is conceivable that cells initiate both CSR and SHM at the same time if AID expression is delayed. All previous and our results are compatible with these scenarios.


    Acknowledgements
 
We are grateful to S. Longerich and T. E. Martin for critical reading of the manuscript and many suggestions, S. Longerich for Fig. 1, and G. Bozek and N. Michael for excellent technical help. This work was supported by NIH grant AI47380. A. L. was supported by a postdoctoral fellowship from the Cancer Research Institute.


    Abbreviations
 
AID—activation-induced deaminase

CDR—complementarity-determining region

CSR—class switch recombination

exo—exonuclease

FWR—framework region

MI—mutation index

MuF—mutator factor

PE—phycoerythrin

PNA—peanut hemagglutinin

pol—polymerase

R/S—ratio of replacement to silent mutations

SHM—somatic hypermutation


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Jacobs, H. and Bross, L. 2001. Towards an understanding of somatic hypermutation. Curr. Opin. Immunol. 13:208.[CrossRef][ISI][Medline]
  2. Storb, U., Klotz, E., Hackett, J., Kage, K., Bozek, G. and Martin, T. E. 1998. A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J. Exp. Med. 188:689.[Abstract/Free Full Text]
  3. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and Honjo, T. 2000. Class switch recombination and somatic hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553.[ISI][Medline]
  4. Revy, P., Muto, T., Levy, Y., Geissman, F., Plebani, A., Sanal, O., Catalan, N., Forveille, M., Dufourcq-Lagelouse, R., Gennery, A., Tezcan, I., Ersoy, F., Kayserili, H., Ugazio, A., Brousse, N., Muramatsu, M., Notarangelo, L., Kinoshita, K., Honjo, T., Fischer, A. and Durandy, A. 2000. Activation-Induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of hyper-IgM syndrome (HIGM2). Cell 102:565.[ISI][Medline]
  5. Goldsby, R., Lawrence, N., Hays, L., Olmsted, E., Chen, X., Singh, M. and Preston, B. 2001. Defective DNA polymerase-delta proofreading causes cancer susceptibility in mice. Nat. Med. 7:638.[CrossRef][ISI][Medline]
  6. Kim, N., Bozek, G., Lo, J. and Storb, U. 1999. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190:21.[Abstract/Free Full Text]
  7. Rogerson, B. 1995. Somatic hypermutation of VHS107 genes is not associated with gene conversion among family members. Int. Immunol. 7:1225.[Abstract]
  8. Michael, N., Martin, T. E., Nicolae, D., Kim, N., Padjen, K., Zhan, P., Nguyen, H., Pinkert, C. and Storb, U. 2002. Effects of sequence and structure on the hypermutability of immunoglobulin genes. Immunity 16:123.[ISI][Medline]
  9. Shen, H., Michael, N., Kim, N. and Storb, U. 2000. The TATA binding protein, c-myc, and surviving genes are not somatically hypermutated, while Ig and BCL-6 genes are hypermutated in human memory B cells. Int. Immunol. 12:1085.[Abstract/Free Full Text]
  10. Hindges, R. and Huebscher, U. 1997. DNA polymerase delta, an essential enzyme for DNA transactions. Biol. Chem. 378:345.[ISI][Medline]
  11. Simon, M., Giot, L. and Faye, G. 1991. The 3' to 5' exonuclease activity located in the polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication. EMBO J. 10:2163.
  12. Blanco, L., Bernad, A. and Salas, M. 1992. Evidence favouring the hypothesis of a conserved 3'–5' exonuclease active site in DNA-dependent DNA polymerases. Gene 112:139.[CrossRef][ISI][Medline]
  13. Morrison, A. and Sugino, A. 1994. The 3'->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol. Gen. Genet. 242:289.[ISI][Medline]
  14. McKean, D., Huppi, K., Bell, M., Staudt, L., Gerhard, W. and Weigert, M. 1984. Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. Proc. Natl Acad. Sci. USA 81:3180.[Abstract]
  15. Di Nola, J. and Neuberger, M. 2002. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glyco sylase. Nature 419:1.[CrossRef][ISI][Medline]
  16. Rada, C., Williams, G., Nilsen, H., Barnes, D., Lindahl, T. and Neuberger, M. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12:1748.[CrossRef][ISI][Medline]
  17. Storb, U. and Stavnezer, J. 2002. Immunoglobulin genes: generating diversity with AID and UNG. Curr. Biol. 12:R725.
  18. Friedberg, E., Walker, G. and Siede, W. 1995. DNA Repair and Mutagenesis. ASM Press, Washington, DC.
  19. Bertocci, B., De Smet, A., Flatter, E., Dahan, A., Bories, J. C., Landreau, C., Weill, J. C. and Reynaud, C. A. 2002. Cutting edge: DNA polymerases mu and lambda are dispensable for Ig gene hypermutation. J. Immunol. 168:3702.[Abstract/Free Full Text]
  20. Esposito, G., Texido, G., Betz, U. A., Gu, H., Muller, W., Klein, U. and Rajewsky, K. 2000. Mice reconstituted with DNA polymerase beta-deficient fetal liver cells are able to mount a T cell-dependent immune response and mutate their Ig genes normally. Proc. Natl Acad. Sci. USA 97:1166.[Abstract/Free Full Text]
  21. Zeng, X., Winter, D., Kasmer, C., Kraemer, K., Lehmann, A. and Gearhart, P. 2001. DNA polymerase eta is an A–T mutator in somatic hypermutation of immunoglobulin variable genes. Nat. Immunol. 2:537.[CrossRef][ISI][Medline]
  22. Zan, H., Komori, A., Li, Z., Cerutti, A., Schaffer, A., Flajnik, M. F., Diaz, M. and Casali, P. 2001. The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation. Immunity 14:643.[CrossRef][ISI][Medline]
  23. Diaz, M., Verkoczy, L. K., Flajnik, M. F. and Klinman, N. 2001. Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase zeta. J. Immunol. 167:327.[Abstract/Free Full Text]
  24. Faili, A., Aoufouchi, S., Flatter, E., Gueranger, Q., Reynaud, C.-A. and Weill, J.-C. 2002. Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota. Nature 419:944.[CrossRef][ISI][Medline]
  25. Rogozin, I., Pavlov, Y., Bebenek, K., Matsuda, T. and Kunkel, T. 2001. Somatic mutation hotspots correlate with DNA polymerase eta error spectrum. Nat. Immunol. 2:530.[CrossRef][ISI][Medline]
  26. Chua, K., Alt, F. and Manis, J. 2002. The function of AID in somatic mutation and class switch recombination: upstream or downstream of DNA breaks. J. Exp. Med. 195:F37.
  27. Liu, Y., Malisan, F., de Bouteiller, O., Guret, C., Lebeque, S., Banchereau, J., Mills, F., Max, E. and Martinez-Valdez, H. 1996. Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation. Immunity 4:241.[ISI][Medline]
  28. Siekevitz, M., Kocks, C., Rajewsky, K. and Dildrop, R. 1987. Analysis of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses. Cell 48:757.[ISI][Medline]
  29. Shan, H., Schlomchik, M. and Weigert, M. 1990. Heavy-chain class switch does not terminate somatic mutation. J. Exp. Med. 172:531.[Abstract]
  30. Petersen, S., Casellas, R., Reina-San-Martin, B., Chen, H., Difilippantonio, M., Wilson, P., Hanitsch, L., Celeste, A., Muramatsu, M., Pilch, D., Redon, C., Ried, T., Bonner, W., Honjo, T., Nussenzweig, M. and Nussenzweig, A. 2001. AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching. Nature 414:660.[CrossRef][ISI][Medline]