Mutagenesis Advance Access published online on May 9, 2008
Mutagenesis, doi:10.1093/mutage/gen019
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Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line
Hubrecht Institute and Cancer Genomics Center, 3584 CT Utrecht, The Netherlands
SN1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.
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N-ethyl-N-nitrosourea (ENU) is one of the strongest chemical mutagens known. In zebrafish this was recognized a long time ago, and since then ENU mutagenesis has effectively been used in numerous forward genetic screens (1–4). In addition to that, target-selected ENU mutagenesis is currently the only available reverse genetics technology to create gene knockouts in both zebrafish and rat, indicating its high efficiency (5–8).
ENU acts by transferring its ethyl group to oxygen and nitrogen atoms of DNA bases. These DNA adducts are not mutations by themselves, but during replication they can mispair, which in the following round of replication can cause a mutation (9
). To protect against this, cells have developed mechanisms to repair alkylation damage. The molecular mechanisms have been studied most extensively for methylation, which will therefore be discussed first. Different types of methylation adducts are repaired by different repair pathways, such as base excision repair (10). The methyl adduct of O6-methyl-guanine (O6-meG) is specifically removed by O6-methyl-guanine methyltransferase (mgmt) (11,12). Additionally, when O6-meG pairs with thymine or cytosine during replication, this is recognized as a mispair by mismatch repair (MMR) enzymes (10,13–16). Those will induce strand excision of the newly synthesized strand until the methylated base pair in an attempt to repair the mismatch. However, since the methylated base is in the template strand of the replicated DNA, it cannot be replaced. Instead, it will continuously be paired with C or T by the polymerase, followed by MMR recognition and excision. This ‘futile cycle’ repair principle will finally result in replication fork stalling and the formation of double-strand breaks (17,18). It leads to apoptosis in MMR-proficient cells and organisms, while MMR mutants are resistant to alkylation-induced death (16–21).
The basic molecular responses to ethylation-induced DNA damage have not been established so far. Different studies using exposure to ENU have resulted in controversial data. Claij et al. (20) found that mouse embryonic stem cells lacking MSH2, a major component of the MMR system, were more resistant to ENU compared to wild-type cells, albeit this difference in survival was less pronounced than for methylation by N-methyl-N'-nitrosoguanidine. These same homozygous mutant cells also displayed a selective growth advantage over heterozygous cells when treated with ENU (19). In contrast to these two reports, no differences were observed between MMR-deficient cells and their wild-type counterparts in response to ENU treatment in two other studies, on human and hamster cells, respectively (22,23). In zebrafish, we observed an increased tolerance to ENU-induced lethality in embryos deficient in msh6 (H. Feitsma, A. Akay and E. Cuppen, in preparation). MSH6 is the MMR component that dimerizes with MSH2 to form a complex that specifically recognizes single-base mismatches and small insertion–deletion loops. Our preliminary results indicate that MMR is very likely to play a role in the recognition of ENU-induced DNA damage in vivo in zebrafish. The molecular mechanism may be analogous to methylation, although it has not been established whether O6-ethyl-guanine (O6-etG) is the principal killing lesion.
Unrecognized alkylated mispairs in MMR-deficient cells that persist in a genome may result in the accumulation of mutations. A higher mutation frequency in MMR-deficient cell lines has indeed been reported for methylation (21) and it was also shown for ethylation, by ENU treatment of mouse embryonic stem cells mutated in msh2 (20).
We used zebrafish mutants deficient in msh6 to test whether a standard zebrafish ENU mutagenesis protocol could result in a higher mutation load in a MMR-deficient background. We observed that neither mutagen-induced lethality nor germ line mutation frequency was different in adult msh6 mutants compared to heterozygous siblings.
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Materials and methods |
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Zebrafish lines
Msh6-mutant fish (hu1811) were obtained by target-selected mutagenesis, and the initial characterization was described elsewhere (H. Feitsma, R. V. Kuiper, J. Korving, I. J. Nijman and E. Cuppen, in preparation). Genotyping was done by polymerase chain reaction (PCR) amplification and resequencing, using exon 10 specific forward (5'-GCTGGTGGCAACTTAAATC-3') and reverse (5'-GCTCAACAGATACTTGCTTTG-3') primers. For the crosses to determine germ line mutation frequency, albino females of the b4 line were used. Those are homozygous for a 4-kb insertion in exon 6 of the zebrafish SLC45A2 gene (E. Wienholds, unpublished data).
ENU mutagenesis
ENU mutagenesis was carried out as described in detail in a ‘Methods in Molecular Biology’ zebrafish volume (E. de Bruijn, E. Cuppen and H. Feitsma, in preparation). ENU was purchased from Sigma (St Louis, MO, USA) in 1 g isopacs, and the appropriate ENU concentrations were carefully prepared using optical density measurement at 238 nm (extinction coefficient = 5830/Mcm) of the ENU stock solution. Fish were treated six times at weekly intervals by bathing them 1 h in ENU in NaPO4 buffer, pH 6.6, followed by a short wash and a recovery phase over night in a dark and quiet room under slight sedation. The next day, they were placed back in their homecages. After four treatments, two females were put in each cage to stimulate spermatogenesis. Animals that became moribund during or after the treatment were sacrificed.
Determination of ENU mutation induction
Clutches fertilized by mutagenized founders were collected and the fertilization rates recorded, after which embryos developed at 28.5°C. Healthy embryos were collected at 5 days post-fertilization and DNA was isolated using standard procedures. For both genotypes, four 96-well plates with embryos from four different founders were used for mutation detection. In total, 33 randomly chosen genomic loci were resequenced. The sequencing procedure was as described previously (6), using a nested PCR and subsequent sequencing reaction. These were ethanol purified and directly run on a 3730XL sequencer (Applied Biosystems, Foster City, CA, USA). Mutation detection was done semi-manually using PolyPhred software and automatically using in-house-developed Perl scripts. Base pair countings were also done automatically with Phred scores of 20 as cut-off. All identified mutations were confirmed in independent PCRs.
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Spontaneous germ line mutation frequency
It was shown previously that zebrafish msh6 knockouts display microsatellite instability (H. Feitsma, R. V. Kuiper, J. Korving, I. J. Nijman and E. Cuppen, in preparation), which is the characteristic form of genomic instability associated with defects in MMR. However, the frequency of spontaneous point mutations has also been shown to be increased in MMR-deficient backgrounds (20,24). This type of mutation
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ENU mutagenesis
Homozygous and heterozygous male msh6 mutant animals of 3 months of age with proven fertility were selected for the mutagenesis experiment. In addition to the standard ENU concentration of 3.3 mM, groups of fish were treated with 2.2 and 4.4 mM ENU to test for changes in tolerance. A fourth group consisted of untreated controls. Six animals of each genotype were used per treatment group. During the treatment period some animals were lost, especially among animals treated with 4.4 mM ENU. In general, no difference in survival between homozygotes and heterozygotes was observed, or if any, the mutants seemed slightly more sensitive, but this was not significant due to small group size (Figure 2).
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From the first week post-treatment, males were crossed with wild-type females. The 4.4 mM ENU-treated animals were in bad condition and were, except for one small clutch from one heterozygous animal, not able to induce egg lays. Otherwise, no significant differences in fertility between genotypes or treatments were observed (Figure 3). The first egg lay of all males of both genotypes was discarded because of potential mosaicism and residual ethylation due to mutagenesis of post-meiotic germ cells (2
), which was also visible by malformations in the embryos. From the second clutches onwards, embryos were collected for DNA isolation.
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ENU-induced germ line mutation frequency
We chose to score mutations in progeny of the 3.3 mM treatment groups only, to be able to obtain a high number of mutations for calculating an appropriate and comparable mutation frequency. Almost 4 million high-quality DNA bases were evaluated and in total 26 mutations were identified for both msh6–/– and msh6+/– animals (Table II). The mutation frequency was therefore similar for MMR-deficient and heterozygous control fish and was found to be around one mutation in 150 000 bp. Since young embryos were used for the frequency determination, it was possible that the frequency was higher than what could be obtained in adults, as embryos with a high hit rate might die during development. To investigate this possibility, F1 adults of a target-selected mutagenesis library obtained from mutagenized wild-type males were resequenced for mutations in a subset of genomic loci. Although these fish were generated in a different experiment, the mutation frequency was found to be virtually identical to that obtained in embryos (Table II).
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When comparing the spectrum of all base pair changes between the different genotype groups, the number of GC to AT mutations tend to be decreased in the germ line of homozygous mutants, which is compensated by an increase of AT to TA mutations (Figure 4). However, these differences were not significant due to the limited number of mutations.
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Discussion |
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We tested the mutagenic activity of ENU in the germ line of male zebrafish deficient in the MMR gene msh6. First, the spontaneous germ line mutation frequency was studied in these mutants using the albino single locus test. Although the number of albinos was small, the mutation frequency was found to be higher than in wild types. However, the observed frequency of one in 5000 embryos in the mutant background is in the same order as the frequency previously reported in wild-type fish (2,25), being around one albino in 10 000 embryos. This indicates that larger amounts of embryos are required to definitively establish an increase of spontaneous mutations in MMR-deficient mutants. As shown for comparison, the ENU-induced germ line mutation frequency has frequently been found to be around one albino in 300 embryos for a good mutagenesis (2,3,8), indicating that the background mutation frequency due to MMR deficiency is one order of magnitude lower. This means that the accumulation of mutations in mutants lacking MMR, and thus presumably the levels of MMR enzymes in wild types, is negligible to the mutation frequency induced by ENU.
Previously, it was reported that MMR-deficient cells are more resistant to alkylation damage. However, the results for ENU are not conclusive, some studies showing increased tolerance in MMR mutants (19,20), while others reported no differences with wild types (22,23). The mechanism of killing of ethylating agents has not been revealed. If alkylation damage recognition mechanisms are similar for methylation and ethylation, then affinities of the polymerase, Mgmt and MMR will probably be different for O6-etG and O6-meG. Another difference is that the level of GC to AT transitions induced by ENU is, at least in mice, relatively low and the level of AT to TA transversions high (26–28), indicating that O6-etG is not the primary mutagenic lesion. In zebrafish, however, GC to AT and AT to TA mutations were found to be equally frequent in a set of positionally cloned mutations from forward screens (4), and GC to AT changes were the most abundant in a relatively unbiased set of sequenced mutations from a reverse genetic screen (7) as well as in samples from wild types and heterozygotes in this study. This indicates that O6-etG is a relevant lesion in zebrafish. However, one would primarily expect an increase in GC to AT transitions in a MMR-deficient environment when O6-etG is the only ENU-induced lesion that is recognized by MMR. In our experiment, a tendency to the opposite effect was observed—a decrease in GC to AT transitions and an increase in AT to TA mutations. Mutations at AT sites were also most frequent in msh2-deficient mouse cells treated with ENU (20). Although the number of identified mutations in both studies was low, these and our results suggest that ENU-induced mutagenesis of A-T base pairs rather than G-C base pairs is suppressed by MMR.
We observed increased survival of msh6-deficient zebrafish embryos compared to wild types after treatment with ENU (H. Feitsma, A. Akay and E. Cuppen, in preparation), indicating that ethylation damage is processed in an MMR-dependent way in vivo in zebrafish. To be able to detect a decreased sensitivity towards ENU, we applied a higher dose of 4.4 mM ENU in addition to the standard 3.3 mM. No differences were observed in survival between heterozygous and homozygous adult mutants. The 4.4-mM mutagenesis concentration was clearly too harsh for the fish. Although some animals survived, only one clutch of eggs was obtained, and most animals became sick during or after the treatments. This high dose may therefore already be too high to reveal a survival difference between MMR-proficient and -deficient fish. A dose–response curve with smaller steps could potentially reveal such differences. Alternatively, the lack of difference in survival between the genotypes can be explained by assuming that the level of DNA damage in adults is relatively low as compared to other cytotoxic effects of ENU, such as ethylation of RNA and toxicity from degradation products (9). The DNA damage caused by alkylation is dependent on replication, and although zebrafish keep proliferating throughout life, the proliferation levels will be much lower in vivo in adult animals than in cultured cells or embryos. The replication-independent toxicity will then be more important for killing, and this is also unrelated to MMR. A third explanation is that the high mutation load is the cause of death of these adult animals. To our opinion this is not very likely, as increased levels of mutations would, differently from its effect in F1 progeny, cause cancer rather than direct lethality. This was not observed, except for one homozygous mutant animal that developed a tumour some time after the treatments.
A higher ENU-induced point mutation frequency has been reported previously for MMR-deficient mouse cells (20). Ethylated base mispairs that do not undergo MMR-dependent apoptosis may result in mutations, and thus result in an increased mutation frequency. In our study, we obtained no indications for an increased mutation frequency in the germ line of msh6-mutant male fish. The observed frequency was one mutation in 150 000 bp in embryos of both homozygous mutants and heterozygous controls, and this was identical to the mutation frequency in adult progeny from treated wild types. Together with the absence of a survival difference, the most obvious explanation for this finding is that, unlike methylation, MMR is not required for ethylation toxicity, and hence, its deletion does not result in an increased mutation frequency. However, since our unpublished findings indicate that there are MMR-dependent effects, other explanations of the current data are possible. The absence of a difference can be explained by the hypothesis that the mutation frequency that is obtained with the highly optimized mutagenesis protocol is the maximum that can be reached in the zebrafish germ line. In favour of this explanation is the fact that highly similar mutation levels are routinely achieved in different experiments in different laboratories. A mutation frequency of one per 100 000–200 000 bp could be the maximum rate that is compatible with a viable genome in general because the maximum mutation frequency that was obtained in Caenorhabditis elegans using ethylmethanesulfonate mutagenesis is one per 109 000 bp (29). Above a certain mutation load, the number of deleterious mutations becomes incompatible with life. In our study, the mutation frequency could be maximized by cell death at two different levels. First, spermatogonial stem cells could be eliminated from the testis, which would result in infertility of males. However, no difference in fertility between the genotypes was observed, and there was no obvious decrease of fertility with increasing doses of ENU. The inability of 4.4 mM-treated animals to induce egg lays was presumably more due to sickness than to infertility, as we know from a previous study that sterile male zebrafish can still induce egg lays without fertilizing them (30). Second, development of fertilized oocytes could be inhibited or arrested due to high numbers of mutations. However, no difference in survival of embryos was found, and the mutation frequency in embryos was similar to that in adult progeny from wild-type mutagenized animals, indicating that loss of progeny during development due to a high mutation load is not likely.
An alternative explanation for the absence of a difference in mutation frequency in MMR-deficient and -proficient backgrounds is that the MMR system in the zebrafish germ line is saturated very rapidly, so that the effect on high-dose ENU mutagenesis is limited. Clearly, for the reported data on mouse msh2-mutant cells, the ENU-induced mutation frequency is of the same order of magnitude as the increase of mutations by loss of MMR alone (ca. 200/106 cells versus ca. 80/106 cells) (20). In fact, almost half of the increase in mutations in ENU-treated mutant cells compared to ENU-treated wild-type cells is attributable to the absence of msh2. This would mean that the levels of ENU-induced damage and of MMR activity are more or less in proportion. We did not determine the frequency of spontaneous point mutations in the germ line of msh6-mutant fish, but according to the albino locus test this is expected to be around 15-fold lower than the frequency that can be induced by ENU. These observations strongly suggest that the capacity of MMR in the zebrafish germ line is negligible compared to the large number of mispairs induced by ENU. However, to firmly establish the role of MMR in zebrafish in this process, more detailed studies on other DNA repair pathways and enzymes are necessary. An example is mgmt, which is highly important in the repair of alkylation damage. Although the Ensembl database (http://www.ensembl.org) gives a zebrafish orthologue for mgmt, there is no data available on whether it is functional and in which tissues. This will be an important direction for future research.
Taken together, survival and germ line mutation frequency of ENU-treated adult male zebrafish are not affected by the absence of MSH6. In contrast to observations in cell culture systems, we did not find increased survival or an increased mutation frequency in vivo. As a result, MMR-deficient backgrounds will not be useful for increasing the efficiency of forward genetic screens or target-selected mutagenesis screens for making gene knockouts in zebrafish.
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Cancer Genomics Center (Nationaal Regie Orgaan Genomics); European Union-funded FP6 Integrated Project ZF-MODELS: LSHG-CT-2003-503496.
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Acknowledgments |
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The authors thank R. van Boxtel for critically reading the manuscript and the Wellcome Trust Sanger Center Danio rerio Sequencing Project for genomic sequence information.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Hubrecht Institute for Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Tel: +31 30 2121969; Fax: +31 30 2516554; Email: e.cuppen{at}niob.knaw.nl
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Received on January 11, 2008; revised on March 20, 2008; accepted on March 20, 2008.
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