ATM Is Not Required in Somatic Hypermutation of VH, but Is Involved in the Introduction of Mutations in the Switch μ Region

Qiang Pan-Hammarström, Shujing Dai, Yaofeng Zhao, Iris F. van Dijk-Härd, Richard A. Gatti, Anne-Lise Børresen-Dale and Lennart Hammarström
J Immunol April 1, 2003, 170 (7) 3707-3716; DOI: https://doi.org/10.4049/jimmunol.170.7.3707

Abstract

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) μ region (up to several hundred base pairs upstream of the Sμ-Sα breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.

Programmed changes in the genomic structure are essential for development of the immune system. During the early stages of T and B lymphocyte differentiation, V(D)J recombination takes place to assemble V exons of the TCR and Ig genes, respectively, giving rise to a large repertoire of lymphocytes, where each lymphocyte expresses a receptor for a given Ag. After activation of the B cells, their Ig genes undergo two types of DNA modification, class switch recombination (CSR)3 and somatic hypermutation (SHM), which further diversify the immune response. In CSR, the C region encoding gene of the μ heavy chain is replaced by a downstream CH gene, resulting in a change from IgM to IgG, IgE, or IgA production, without changing the specificity of the Ab. SHM, on the other hand, leads to accumulation of mutations in the V genes and, when coupled with selection, results in an increase in the affinity for the Ag.

Like V(D)J recombination, CSR involves DNA deletion by a mechanism by which intervening sequences are excised as circular DNA (1). Moreover, CSR resembles V(D)J recombination in that a DNA double-strand break (DSB) intermediate appears to be part of this reaction (2). CSR is however distinct from V(D)J recombination as it occurs later in B cell development, independent of RAG1 and RAG2, and it is region-specific rather than site-specific. Recent studies have shown that CSR requires proteins in the nonhomologous end joining (NHEJ) pathway. These include DNA-PKcs, Ku70 and Ku80 (3, 4, 5), suggesting that CSR is executed by the general repair machinery that is also involved in the V(D)J recombination process and repair of gamma irradiation-induced DNA damage. SHM and CSR can occur independently in the germinal center, but probably share some of the proteins/pathways involved, as frequent mutations are observed in the vicinity of the CSR breakpoints. Furthermore, large deletions have been reported in the V region in selected clones, and two studies have shown that the germinal center-specific activation-induced cytidine deaminase (AID) plays a key role in controlling both processes (6, 7). Recently, Honjo and coworkers (8) have also demonstrated AID-dependent induction of SHM-like point mutations in the Sμ region in mouse lymphocytes, further supporting the idea that AID may mediate a common step in both SHM and CSR.

Ataxia-telangiectasia (A-T) is a rare, complex, multisystem disorder characterized by cerebellar degeneration with ataxia, ocular apraxia, ocular and cutaneous telangiectasias, radiosensitivity, chromosomal instability and cancer predisposition (for review see Refs. 9 and 10). The ATM gene (mutated in A-T) was previously identified as a member of a family of phosphatidylinositol 3-kinase-related genes and is one of the master controllers of the networks involved in cell cycle control and response to DNA damage.

A-T is also recognized as a primary immunodeficiency syndrome involving both humoral and cellular immunity and recurrent infections, primarily affecting the respiratory tract (10, 11). IgA deficiency has been observed in 60–80% of patients, and a subgroup suffers from concomitant IgG subclass deficiency, suggesting a defect in switching and subsequent production of downstream Ig isotypes as an underlying cause of the susceptibility to infections (9, 10). Defects in the Ab repertoire, indicative of restricted use of V genes and/or affinity maturation of the Abs produced, have also been suggested in A-T patients, and a number of studies (12, 13, 14) have demonstrated a reduced production of Abs against selected pathogens. Furthermore, proneness to infections is not directly correlated to Ig class or subclass levels (14), suggesting a lack of generation of specific Abs or lack of affinity maturation in A-T patients.

We have recently shown that the CSR junctions in cells from A-T patients are aberrant, supporting a role of ATM in the final steps of CSR, including DNA end modification, repair, and joining (15). The junctions from A-T patients are characterized by a strong dependence on short sequence homologies and are devoid of normally occurring mutations/insertions around the breakpoint. The latter feature may suggest a direct role for ATM in the SHM process. In this report we therefore compared mutation patterns in the V and Sμ regions in cells from both normal individuals and A-T patients.

Materials and Methods

Patients

The study included 14 A-T patients (11 Swedish patients, one Danish patient, one Finnish patient, and one Norwegian patient). Clinical examination of these patients was undertaken by the respective Pediatric Departments in the Nordic countries, and the clinical and biochemical data have been published previously (16) (for further details, see ATbase: www.cnt.ki.se/ATbase). All patients have been screened for ATM mutations using a single-strand conformation polymorphism assay (17) and/or a protein truncation test (18, 19).

A-T cell lines are EBV-transformed lymphoblastoid cell lines, derived from patients with A-T, under human subjects protection committee-approved protocols. EBV-transformed human B cell lines used for controls were provided by Prof. I. Ernberg (Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden). The cells were grown in RPMI 1640 with 15% FBS and penicillin/streptomycin/glutamine (1%; Life Technologies, Gaithersburg, MD) in a humidified CO2 incubator at 37°C.

RNA isolation and PCR amplification of VH3-JH and VH3-Cγ transcripts

Total RNA was extracted from PBL using RNeasy RNA purification kits (Qiagen, Hilden, Germany), and first-strand cDNA synthesis was performed with Cγ (CγA, 5′-GTCCTTGACCAGGCAGCCCAG-3′) or Cμ-specific primers (CμA, 5′-GAGGCAGCTCAGCAATC) using a cDNA synthesis kit (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. The primers used for amplification of VH3-JH transcripts were VH3 consensus (5′-aatctagaGGTGCAGCT GGTGGAGTC-3′) and JH consensus (5′-cagtcgacCCTGAGGAGACGGTGAC-3′). The primers used for amplification of VH3-Cγ transcripts were VH3 consensus and CγB (5′-cagtcgacAAGACCGATGGGCCCTTGGTGG-3′). The oligonucleotides contained a restriction site (underlined; XbaI in the VH3 consensus primer and SalI in the JH consensus and CγB primers) for directional cloning of the PCR products. Amplification was performed in 40 cycles, each cycle consisting of 94°C for 50 s, 62°C for 1 min, and 72°C for 1 min. A high fidelity Vent DNA polymerase (New England Biolabs, Hertfordshire, U.K.) was used in all PCR amplifications of the V region transcripts.

Analysis of the VH-JH and VH-Cγ clones

The PCR products were purified and cloned into the Bluescript II KS+ vector (Stratagene, La Jolla, CA) and transformed into JM 109 competent cells. The resulting clones were screened by PCR amplification (VH3 consensus/JH consensus or VH3 consensus/CγB), and the positive clones were sequenced by an automated fluorescent sequencer (AB1, 373A-Stretch, PerkinElmer; from Cybergene, Huddinge, Sweden) using a BigDye terminator cycle sequencing kit (PerkinElmer). Sequence analysis was performed using DNAPLOT software (V base, http://www.mrc-cpe.cam.ac.uk/imtdoc/public/INTRO.html) and IMGT/V-QUEST (http://imgt.cines.fr) (20) to align the VH-JH and VH-CγB sequences to their closest germline V, D, and J segment counterparts. The Ig V(D)J junctional sequences were analyzed by the IMGT/JunctionAnalysis tool, available at http://imgt.cines.fr.

Amplification of Sμ-Sα fragments and the germline Sμ region

The amplification of Sμ-Sα fragments from in vivo switched cells was performed as described previously using Taq DNA polymerase (15, 21). Briefly, two pairs of Sμ- and Sα-specific primers were used in a nested PCR assay. The number of Sμ-Sα fragments was determined from 10 PCR reactions run in parallel using DNA from one individual and represents random amplification of in vivo switched clones. Patient samples were always run at the same time as the control samples. A human IgA1-producing cell line (313) was also run in parallel to assess the fidelity of the nested PCR reaction used to amplify Sμ-Sα fragments from the patient and control samples (15). On the average, 6.5 ± 3.1 switch fragments were randomly selected from each control individual for cloning and sequencing. In the A-T patients, all the switch fragments (on the average, 4.4 ± 2.5 from each patient) were subjected to cloning and sequencing, as fewer switch fragments could be amplified from A-T cells.

A long PCR kit (Expand Long Template PCR System Kit; Roche Diagnostics Scandinavia, Bromma, Sweden) was employed to amplify the full-length germline Sμ region. This system uses an enzyme mixture containing Taq DNA polymerase and proof-reading Pwo DNA polymerase. Genomic DNA from one individual with a known germline Sμ sequence and DNA from BAC66R4C11, which contains a bovine Ig-encoding gene (22) were used to assess the fidelity of the long PCR reaction. Independent PCR products were cloned, and mutations were shown to be introduced at a rate of 2/10,000 nucleotides in the 20 clones analyzed. Sμ-specific primers Hu-SμLs (5′-GGGGACCTGCTCATTTTTATCACA) and Hu-SμLas (5′-GAGGACCCGCAGGACAAAAGAGAA) were chosen from the region flanking the Sμ repetitive sequences. Amplification was first performed in 10 cycles, where each cycle consisted of 92°C for 10 s, 64°C for 30 s, and 68°C for 4 min, and subsequently in 20 cycles with the same conditions, except that the elongation time was extended for 10 s for each new cycle. PCR products (4.4 kb) were gel-purified and subsequently used for direct sequencing. The sequencing primer Sμ5 (23) is located 235 bp downstream of the Hu-SμLs primer. The first 450 bp of the sequence was analyzed, covering the region where all the Sμ-Sα breakpoints are located.

Results

Analysis of mutations in VH-JH transcripts in A-T cell lines

To investigate the functional role of ATM in the SHM process, we first studied the V(D)J regions in EBV-transformed cell lines from four A-T patients and six controls. The cDNA synthesis was primed either by a Cγ-specific (CγA) or a Cμ-specific (CμA) oligonucleotide, and the entire VH regions were PCR amplified by VH3 consensus and JH consensus primers. The IgM-positive A-T cell line MGR showed a germline VH region sequence, and the three IgG-positive A-T cell lines showed one, four, and five mutations, respectively, whereas the IgM-positive (n = 2) and IgG-positive (n = 4) cell lines from normal blood donors, respectively, showed four and 24 mutations, on the average (data not shown). This may suggest a lower frequency of hypermutation in A-T patients; however, the number of clones was too low to allow a reliable statistical analysis.

Analysis of mutations in VH-Cγ transcripts in A-T patients

We subsequently sequenced PCR-amplified Ig VH transcripts derived from PBL from A-T patients. RNA samples from seven A-T patients and seven age-matched controls (range, 5–31 years) were prepared, and single-strand cDNA synthesis was performed using the CγA primer. Our first approach was to analyze mutations in the transcription of the VH3–23 gene, a member of the VH3 family expressed in 4–10% of human B cells (24). However, we could only generate a few clones from the A-T patients even after several rounds of PCR amplification/cloning, which could be due to the limited amounts of RNA used for cDNA synthesis (only 0.3–1 μg of RNA was available from each patient). We therefore employed a VH3 consensus primer and a Cγ-specific primer (CγB), and the resulting PCR products were cloned. The same pair of primers was used for screening, and positive clones were verified by sequencing. In total, 52 and 48 distinct VH-Cγ clones were generated from B cells of patients and controls, respectively. The majority of these clones contained VH region genes that belonged to the VH3 family, and a few contained VH1 (VH1–18 and VH1–69) or VH5 (VH5–51) genes. Most of the VH gene sequences from the patients were mutated (2–31 bp substitutions/clone), and only ∼4% of the VH region genes exhibited unmutated sequences (0–1 bp substitution/clone). Overall, the frequency of mutations in the VH genes derived from A-T patients varied from 3.8 to 6.4%, which is similar to that found in the age-matched control subjects (Table I⇓). The ratio of replacement vs silent mutations (R/S) was also similar in the two groups, arguing against abnormalities in the Ab selection process in our A-T patients.

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Table I.

Mutations in VH3-Cγ transcripts from PBL from A–T patients and controls

The pattern of mutations (727 mutations in total, 14.0/VH) observed in the A-T patients was also broadly similar to that found in the normal controls (619 mutations in total, 12.9/VH), with major hotspots of mutation (AGT and AGC triplets, at codons Ser31, Ser52, or Ser82), conforming to the previously described hotspot consensus RGYW motif (25, 26) (Fig. 1⇓). The mutations showed a preference for transitions (purine (A,G) to purine, or pyrimidine (C,T) to pyrimidine; 57%) rather than transversions (where purine is replaced by a pyrimidine or reverse) and a DNA strand polarity, as manifested by the increased targeting for mutation of A residues on the coding strand over T residues (see Table A in supplementary data).4 However, no differences were found in the nature of base pair substitutions between A-T patients and normal controls (see Table A in supplementary data).

FIGURE 1.
FIGURE 1.

The distribution of mutations in VH-Cγ sequences in PBL of normal individuals and patients with A-T. Sequences were determined from the framework region 1 to the beginning of Cγ region, and mutations between codons 9 and 94 of the VH mature polypeptide were computed. A, Mutations in controls. B, Mutations in A-T patients.

VH3–7 and VH3–23 genes were used frequently both by A-T patients and age-matched controls (see Table B in supplementary data). VH3–11, VH3–30.3, and VH3–33 genes were rearranged more frequently in cells from A-T patients, whereas the VH3–74 gene was only seen in control clones (9.5%). When combined with the data from cell lines, it appears that the VH3 genes in the 3′ half of the VH locus were preferentially used in A-T patients (88.6%) compared with controls (67.4%; by χ2 test, p < 0.05). However, overall, no significant differences were observed between the controls and patients in terms of individual VH3 gene usage, except for the VH3–74 gene, which was significantly less often used by cells from A-T patients (by χ2 test, p < 0.05).

Characteristics of the complementarity-determining region 3 (CDR3) in B cells from A-T patients

Diversity in the CDR3 in clones derived from A-T patients was also analyzed. The CDR3 comprises contributions from both the VH, D, and JH gene segments and nucleotides added by TdT. All the clones were in-frame rearrangements. The average length of the CDR3 was 12.1 ± 3.9 aa in the A-T clones and 12.6 ± 5.4 aa in control clones. Most clones (50 of 52 A-T clones and 42 of 48 of control clones) had N regions at either the VH-D or the D-JH junctions. There were no significant differences in the length of the N regions observed between patients and controls (see Table C in supplementary data). The presence of possible P nucleotides, i.e., nucleotides that were originally complementary in the dsDNA and therefore form short DNA palindromes, was observed in 18 and 14 clones from patients and controls, respectively. There was a preferential use of segments within the D2 and D3 families in clones from both A-T patients and controls. Furthermore, although there were significantly more clones from A-T patients using the JH4 segment (p < 0.001) and fewer containing the JH5 segment (p < 0.05; see Table B in supplementary data), the V(D)J coding joints were largely normal.

Mutations in the Sμ region in normal controls and A-T patients

We have previously analyzed the sequence of Sμ-Sα junctions (±15 bp) from in vivo switched cells in both normal healthy individuals and A-T patients. Ninety-five percent of the Sμ-Sα junctions from A-T patients display short sequence homologies (microhomology; ≥1 bp), i.e., nucleotides that are shared by both the Sμ and Sα regions. Moreover, more than half of the junctions (61%) exhibit a perfect-matched microhomology of ≥4 bp (15). Lack of mutations or insertions is a second feature of the switch junctions in cells from A-T patients (15). We now extended the sequence analysis to the Sμ region in the generated switch fragments (110 from normal individual and 44 from A-T patients). To exclude polymorphisms, germline Sμ regions (4.4 kb) were amplified from all normal individuals and A-T patients who contributed to the switch fragment sequences using a high fidelity enzyme mixture. Part of the germline Sμ region (encompassing 450 bp) was analyzed, covering the region where all the Sμ breakpoints were located. There were two nucleotides at positions 301 and 319 in the Sμ region that differed from the published sequence (27), and as these changes (a deletion of A and an insertion of G) were present in all individuals analyzed, it suggests a PCR error or, alternatively, a rare polymorphism in the previously described sequence. Several additional polymorphisms in this region were observed in two normal individuals and two A-T patients, and those were excluded from the mutation analysis. The Sμ sequences, starting 15 bp upstream from the breakpoints, were mutated in about half the control and A-T switch fragments (Table II⇓). The mutations were distributed rather evenly among the mutated A-T switch fragments, most of which carried one or two mutations, whereas a few (n = 3) showed three or four mutations per switch fragment. In controls, most mutated switch fragments also carried one to four mutations, but a marked proportion (14%, n = 8) carried 5–10 bp mutations/switch fragment (for details see Table III⇓⇓). In total, 130 point mutations were found in the Sμ sequences (upstream of the switch junctions) in normal controls (n = 17) in the 19,335 bp sequences analyzed, a frequency of 6.7/1,000 bp (Table II⇓). This frequency is much higher than the PCR error rate in an IgA-producing cell line run in parallel as a control (0.9/1,000 bp) (15), suggesting that point mutations accumulate in the Sμ region in normal B cells undergoing switching. In A-T patients (n = 10), only 36 mutations were found in the 11,993 bp sequences analyzed, a frequency of 3.0/1,000 bp (Table II⇓). This frequency is significantly less than that in control subjects (by Mann-Whitney’s test, p < 0.05), but clearly above the PCR error rate.

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Table II.

Mutations in the Sμ region (upstream of the switch junctions) in normal controls and A–T patients

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Table III.

Characterization of the individual mutations in the Sμ region (upstream of the switch junctions) in normal controls and A-T patients

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Table IIIA.

Continued

Mutations were often associated with the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif in both A-T patients and controls (67 and 71%, respectively; Fig. 2⇓ and Table III⇑⇑). Although this motif is frequently occurring in the Sμ region, the percentage of the mutations is still significantly higher than expected by chance (51%; by χ2 test, p < 0.001 for controls). The most often targeted motifs in the part of the Sμ region analyzed are AGCT, AGTT, AACT, and, less frequently, GGTT. It is worth noting that the last T in these motifs was often mutated in A-T patients (11 of the 24 mutations present in these motifs), whereas they were seldom mutated in controls (6 of 88; by χ2 test, p < 0.001; Fig. 2⇓). The clustering of mutations at the T nucleotides in these motifs in A-T patients could not explained by sampling bias, as this was observed in distinct switch fragments from multiple individuals (Fig. 2⇓). Furthermore, the general pattern of base substitutions was different from that in controls, with more at A/T sites (56 vs 34%; by χ2 test, p < 0.05) and a strong preference for transitions (86 vs 58%; by χ2 test, p < 0.01; Table IV⇓). Thus, an ATM-controlled mechanism seems to be involved, but is not essential for introducing mutations in the Sμ region, and in the absence of ATM, pathways controlled by other proteins dominate, skewing the normal base substitution pattern.

FIGURE 2.
FIGURE 2.

Distribution of mutations in the Sμ region. The germline Sμ sequence (position 51–400 in Ref. 27 ) is shown by uppercase letters. Mutations in the Sμ region in A-T patients and controls are shown above and below the germline sequences, respectively, by lowercase letters. The sequences that belong to RGYW/WRCY motifs are underlined. The origins of T→C mutations in A-T patients are indicated within parentheses (sample name-clone name).

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Table IV.

Nature of base substitutions in the switch μ regiona

The human S region contains a large number of palindromic sequences that can create secondary ssDNA structures. As previously demonstrated in Xenopus (28), CSR often occurs at positions (microsites) where a ssDNA folding analysis predicts a transition from a stem to a loop structure. We have also mapped mutations in the secondary structures of the human Sμ regions (software available at http://bioinfo.math.rpi.edu/∼mfold/dna/form1.cgi). About one-third of the Sμ mutations found in A-T patients could be mapped to predicted microsites, a frequency not significantly different from that in controls (data not shown). Thus, the altered mutation pattern in the S region in A-T patients is unlikely to be due to abnormalities in recognition of the targeting structure and/or a DNA cleavage step of CSR.

Discussion

CSR and SHM occur primarily in germinal center B cells, are linked to transcription, and are impaired by deficiency of AID, suggesting a mechanistic link between the two processes (for review, see Ref. 29). ATM is essential for repair of the DNA DSBs induced by gamma irradiation and is involved in homologous end-joining pathways (30). We have recently shown that switch junctions are aberrant in cells from A-T patients and have suggested a potential role for ATM in the NHEJ pathway in CSR (15). As lack of mutations around the switch junctions was a unique feature in A-T cells, we hypothesized that CSR might share an ATM-dependent end joining/repair pathway with SHM. Although there was a trend for fewer mutations in the V regions in A-T cell lines, results from IgG-positive cells showed that there were no differences between normal donors and A-T patients in the proportion of V genes that showed hypermutation, the frequency and distribution of mutations, or the nature of base pair substitutions. ATM is thus unlikely to be involved or is redundant in the SHM process.

Honjo and co-workers (8) have recently reported that murine B cells stimulated with LPS/IL-4 accumulate SHM-like point mutations in the Sμ region, a process dependent on AID. In this study we show that mutations could also be found upstream of the switch junctions, in the Sμ region derived from normal human B cells. Although we would assume that these sequences are derived from in vivo switched lymphocytes, this cannot be established with absolute certainty. The frequency of the mutations in the S and V regions in our study is markedly higher than that in LPS/IL-4 stimulated mouse cells (8), which could be due to differences in the nature of the stimuli acting on cells undergoing switching in vitro and in vivo and the population of cells studied. The mutations in the S regions (upstream of the breakpoints) show a similar spectrum as those in V regions and often occurring in the predicted SHM hotspots (Fig. 2⇑ and Table V⇓). Thus, these data seem to support the hypothesis that a similar cellular machinery, involving AID, may initiate SHM and CSR by producing lesions in both the V genes and S regions (31). In A-T patients a number of mutations could also be observed in the Sμ region, upstream of the breakpoints. However, these occur with about half the frequency of that in controls and, more interestingly, were almost only due to transitions and biased to mutations of A/T nucleotides (Tables IV⇑ and V⇓). This argues against the possibility that mutations in the Sμ regions (upstream of the breakpoints) are produced by the same machinery used in the SHM pathway. At least part of the mutations in the S region seem to be generated by an ATM-dependent pathway, involving factors that act as G/C mutators and/or are responsible for transversions. Alternatively, diminished CSR in A-T patients might contribute to the reduction of mutations in the S region. However, this cannot explain the bias for the selective base pair substitutions.

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Table V.

Comparison of mutations in the V and S regions from normal individuals and A–T patients

It is also important to note that in normal controls, mutation patterns in the V and Sμ regions (upstream of the breakpoints) are clearly different from those at or close to the switch breakpoints (Table V⇑ and Fig. A in supplementary data). The latter showed a strong bias toward mutations of G/C nucleotides. Transversions were favored; furthermore, they occurred at a rather high frequency (20.0/1000 bp). In the absence of ATM, the mutation frequency at or close to the switch breakpoints drops from 20.0/1000 bp to 3.0/1000 bp, a frequency also seen upstream of the switch breakpoints in these patients (Table V⇑). It is possible that mutations at or close to the switch breakpoints were mainly generated during the final repair step(s) in CSR, which is ATM dependent, whereas the mutations away from the breakpoints were caused by an earlier reaction(s) in CSR, which may share some of the factors with SHM.

Several possible mechanisms can be suggested for how ATM may affect the switching process. First, ATM might be involved in chromosomal accessibility, as it is associated with chromatin and the nuclear matrix (32). Furthermore, the rapid dephosphorylation of histone H1 in response to ionizing radiation is dependent on ATM (33). However as SHM and V(D)J recombination are largely normal in A-T patients, the proposed role of ATM in chromosomal accessibility has to be specific for CSR. Second, ATM might be involved in the end joining/repair machinery in CSR, possibly through phosphorylation of or interaction with additional factors that have been implicated in CSR, such as the Ku heterodimer (4, 5), mismatch repair enzymes (34, 35), and the hMre11/hRad50/p95(nibrin) complex (36).

It has previously been suggested that AID is primarily involved in the generation of mutations at G/C nucleotides in the V region (37), and quite recently Neuberger and co-workers (38) have shown that the expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG. They also proposed a DNA deamination model of Ig gene diversification where SHM, CSR, and gene conversion are all initiated by AID-mediated deamination of dC residues in the Ig locus. Later, depending on which way the initiating dU/dG lesion is resolved, it will result in a different outcome, i.e., SHM, CSR, or gene conversion (38). Based on this model, the different impacts of deficiency of mismatch repair enzymes (39), DNA-PKcs (3, 40), or ATM on CSR and SHM may thus reflect differences in the pathways of break resolution.

Experiments in SV40-transformed fibroblast cell lines from two A-T patients have previously shown normal levels of V(D)J recombination using an extrachromosomal substrate assay (41). We have now shown that the quality of the Ig V(D)J-coding joints from A-T cells are also largely normal. However, both A-T patients (42) and ATM-deficient mice (43, 44) are prone to lymphoid malignancies that harbor translocations involving V(D)J region genes. Furthermore, ATM-deficient mice no longer develop tumors with such translocations when the V(D)J recombination is blocked by mutations in the RAG1 gene (45). Moreover, a recent study has provided direct evidence that both ATM and a product of ATM kinase activity, Ser18-phosphorylated p53, are recruited to DSBs associated with V(D)J recombination (46). ATM may thus still be involved, but dispensable, in normal V(D)J recombination, but it is required in protection against tumor development caused by aberrant V(D)J recombination. The biased usage of downstream of VH3 genes and upstream JH genes in A-T patients may also suggest a potential role for ATM in receptor revision, where secondary Ig gene rearrangements occur in mature B cells in the periphery (47).

In summary, V(D)J recombination, SHM, and CSR may share some of the molecular mechanisms involved, but are differentially regulated. ATM seems dispensable in the SHM and V(D)J recombination processes, but is clearly involved in the end joining-repair machinery in CSR.

Footnotes

  • ↵1 This work was supported by the Swedish Research Council, SSFM and funds from the Karolinska Institute.

  • ↵2 Address correspondence and reprint requests to Dr. Qiang Pan-Hammarström, Clinical Immunology, IMPI, Karolinska Institute at Huddinge Hospital, SE-141 86 Stockholm, Sweden. E-mail address: qipa{at}biosci.ki.se

  • ↵3 Abbreviations used in this paper: CSR, class switch recombination; AID, activation-induced cytidine deaminase; A-T, ataxia-telangiectasia; ATM, mutated in A-T; CDR3, complementarity-determining region 3; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DSB, DNA double-strand break; M/R/N, hMre11/hRad50/p95(nibrin); NHEJ, nonhomologous end joining; S, switch; SHM, somatic hypermutation.

  • ↵4 The on-line version of this article contains supplemental material

  • Received June 5, 2002.
  • Accepted December 6, 2002.

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