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
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Abstract |
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Keywords: class switch recombination, polymerase exonuclease, somatic hypermutation
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Introduction |
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Mice homozygous for a pol (pol
) exonuclease (exo) inactivating point mutation were generated by Goldsby et al. (5). In order to determine if pol
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.
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Methods |
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Immunization of pol 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 2030 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: CD19FITC, CD19biotin followed by streptavidinCyChrome, B220biotin followed by streptavidinCyChrome, CD43phycoerythrin (PE), CD25PE, IgMPE, IgDa and IgDbFITC (both allotypes), CD3PE, GL7FITC (all from PharMingen, San Diego, CA); peanut agglutinin (PNA)FITC (Sigma, St Louis, MO); PNAbiotin (Vector Laboratories, Burlingame, CA) followed by streptavidinPE (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 primer GXCH1 (5'-CCAGGGGCCAGTGGATAGAC-3') which primes all
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.
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Results |
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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 constant regions. In all three pol
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 102, 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
exo/ mice is normal.
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In the 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
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 exo/ mice; thus, the SHM process continues over several cell generations also in these exo deficient mice. Multiple pedigrees in all three pol
genotypes suggest that SHM and CSR are intermingled processes (see Discussion).
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Discussion |
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Inactivation of the 3'5' exo by one of several point mutations in the exo domains of pol inhibits its proofreading capacity, while retaining the pol activity (1113). Mice with the homozygous knock-in of a pol
exo mutation produce normal levels of pol
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 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
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 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
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
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 12 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 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
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 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,
, µ or
, 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
has been shown to lead to an altered mutation spectrum (21). Inhibition of pol
decreases SHM levels (22,23). Pol
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
exo mutation on SHM could be due to the following: (i) normally pol
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
is involved in SHM, but the exo is prevented from proofreading, and (iii) pol
is not creating somatic mutation.
The possibility that pol 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
increases its fidelity 130- to 1900-fold (13). It would be expected that with exo pol
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 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
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
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 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
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 Weigerts laboratory (29). It is also interesting to note that in most of the pedigrees the 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 VDJCµ 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.
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Acknowledgements |
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Abbreviations |
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CDRcomplementarity-determining region
CSRclass switch recombination
exoexonuclease
FWRframework region
MImutation index
MuFmutator factor
PEphycoerythrin
PNApeanut hemagglutinin
polpolymerase
R/Sratio of replacement to silent mutations
SHMsomatic hypermutation
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References |
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