Mutagenesis in PMS2- and MSH2-deficient mice indicates differential protection from transversions and frameshifts
Susan E. Andrew4,
Xiaoxin S. Xu1,
Agnes Baross-Francis2,
Latha Narayanan1,
Kate Milhausen2,
R.Michael Liskay3,
Frank R. Jirik2 and
Peter M. Glazer1
Department of Medical Genetics, University of Alberta, Edmonton, AB T6G 2H7 Canada,
1 Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, Boyer Center Room 354, 295 Congress Avenue, New Haven, CT 06536-0812, USA,
2 Centre for Molecular Medicine and Therapeutics and Department of Medicine, University of British Columbia, Vancouver, BC V5Z 4H4 Canada and
3 Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR 97201-3098, USA
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Abstract
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DNA mismatch repair (MMR) deficiency leads to an increased mutation frequency and a predisposition to neoplasia. `Knockout' mice deficient in the MMR proteins Msh2 and Pms2 crossed with mutation detection reporter (supF, lacI and cII) transgenic mice have been used to facilitate a comparison of the changes in mutation frequency and spectra. We find that the mutation frequency was consistently higher in Msh2-deficient mice than Pms2-deficient mice. The lacI target gene, which is highly sensitive to point mutations, demonstrated that both Msh2- and Pms2-deficient mice accumulate transition mutations as the predominant mutation. However, when compared with Msh2/ mice, lacI and cII mutants from Pms2-deficient mice revealed an increased proportion of +/1 bp frameshift mutations and a corresponding decrease in transversion mutations. The supF target gene, which is sensitive to frameshift mutations, and the cII target gene revealed a strong tendency for 1 bp deletions over +1 bp insertions in Msh2/ compared with Pms2/ mice. These data indicate that Msh2 and Pms2 deficiency have subtle but differing effects on mutation avoidance which may contribute to the differences in tumor spectra observed in the two `knockout' mouse models. These variances in mutation accumulation may also play a role, in part, in the differences seen in prevalence of MSH2 and PMS2 germline mutations in hereditary non-polyposis colorectal cancer patients.
Abbreviations: MMR, mismatch repair; HNPCC, hereditary non-polyposis colorectal cancer syndrome.
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Introduction
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The current mammalian model of DNA mismatch repair (MMR) implicates MSH2 (1,2) as being critical for the initial recognition of mismatches. Heterodimers formed between MSH2 and either MSH3 or MSH6/GTBP (3,4) are necessary for repair of single base mismatches or small insertions or deletions (5). Recruitment of a second heterodimer, made up of PMS2 (6) and MLH1 (7,8), is thought to be necessary for subsequent excision and resynthesis necessary to repair the region containing the mismatched base(s) (2).
Approximately 70% of individuals with hereditary non-polyposis colorectal cancer syndrome (HNPCC) carry germline mutations in one of either MSH2, MLH1, PMS1 or PMS2 (9) and are characterized by a high incidence of colon cancer at an early age, as well as an increased predisposition to tumors of the endometrium, gastrointestinal tract and skin (10,11). Loss of the wild-type MMR allele is present in tumors arising in these individuals. Most of all HNPCC mutations that have been identified occur in the MSH2 and MLH1 genes. Interestingly, PMS1 and PMS2 appear to be mutated only rarely (6,12), suggesting that they likely have overlapping functions with other compensating molecules. PMS1 has no known biochemical role and Pms1 knockout mice are not prone to tumorigenesis (13), so the role of PMS1 in mutation avoidance may be restricted to specific sequences and may be tissue and/or cell type specific. In the absence of MMR, tumor development or progression is thought to be accelerated due to the resulting hypermutability, with mutations accumulating in key tumor suppressor genes, oncogenes and other genes involved in growth control (12).
Mice lacking Pms2 and Msh2 have provided a model system to study the roles of the specific MMR proteins in DNA repair and cancer (1316). Mice deficient in any one of the MMR proteins, except Pms1, are characterized by increased tumor development after 2 months of age (13). The presence and early onset of tumors is further supporting evidence of the vital role Pms2 and Msh2 are assumed to play in maintaining a stable genome and suggests that both molecules play an important role in murine tumorigenesis. However, mice lacking various components of the MMR complex all demonstrate differences with respect to tumor spectrum (13,17,18) consistent with differing functional roles of specific MMR molecules or possibly reflecting subtle differences in residual repair or partial redundancy from MMR gene paralogs. Msh2/ mice develop sarcomas, lymphomas, skin neoplasms and intestinal tumours (13,1618), whereas Pms2/ mice develop only lymphomas and sarcomas (13,14). The various MMR knockouts have been constructed in two different strains of 129 (Ola and Sv) which may account for some of the differences in tumor spectra, however, the effect of substrain variability on MMR is likely subtle compared with the MMR deficiency itself (1316,1921).
Mutation detection transgenic mice are invaluable for investigating the role of MMR deficiency at the single gene level in vivo. The incorporation of a retrievable reporter gene such as lacI, supF or cII into a murine host makes possible the collection of tissue-specific mutation frequency data and mutant recovery. Sequencing of the recovered mutant genes provides the opportunity to determine gene-specific mutation spectra on different MMR-deficient backgrounds as mutations within the reporter genes may reflect those occurring within endogenous genes.
Previous data from Msh2//lacI and Pms2//supF mice demonstrated dramatically different levels of mutation frequency and differences in mutation spectra (22,23). Pms2-deficient mice had far greater supF mutation frequency elevations than those observed within the lacI genes rescued from Msh2//lacI mice. As Msh2 and Pms2 are thought to interact in a multiprotein complex and both are thought to be essential for functional MMR repair, the quantitative and qualitative differences in the two `knockout' mice required further investigation. It was possible that the mutation differences reflected the different nature of the two target genes used in the different studies. Alternatively, the consequences of losing Msh2 or Pms2 may not have been equivalent in terms of resulting mutability, with loss of Pms2, for example, leading to greater instability.
In order to determine the potential differences of Msh2 and Pms2 deficiency on DNA mutation, Pms2/ and Msh2/ mice were bred to both supF- and lacI-containing mice, thus allowing mutation frequencies and spectra to be directly compared. The cII gene of phage carrying the supF reporter was also used as a third target gene to compare mutation frequencies between Pms2 and Msh2 null mice. The results from all reporter genes suggest that mutation frequencies may be higher in animals lacking Msh2 than in those deficient in Pms2. Pms2/ mice had a greater proportion of frameshift mutations and fewer transversions than Msh2/ mice. Furthermore, while both +1 and 1 bp frameshifts were recovered from Pms2/ mice, frameshift mutations obtained from Msh2/ mice were predominantly deletions. These differences between Msh2- and Pms2-deficient mice suggest that these two proteins have different roles in DNA damage or mispair identification and repair. These differences may account for variations in cancer predisposition between the `knockout' mice and suggest a plausible explanation for the lower incidence of Pms2 germline mutations in HNPCC patients (24).
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Materials and methods
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Transgenic mice and genotype testing
Pms2-deficient mice created through gene targeting in embryonic stem cells (14) were crossed with transgenic lines carrying either a lacI (25) or a supF mutational reporter gene (26,27) to obtain Pms2+/+/lacI, Pms2+//lacI and Pms2//lacI and Pms2+/+/supF, Pms2+//supF and Pms2//supF offspring, respectively. Similarly, Msh2-deficient mice (16) were crossed with mice transgenic for the lacI or supF reporter genes. The Msh2/ mice are on a 129/Ola genetic background, the Pms2/ are 129/Sv, the lacI mice are Balb/cJ and the supF mice are C57BL/6. The MMR heterozygous mice were bred with reporter transgenic mice and the MMR-deficient, transgene-carrying offspring were used for the following experiments. Pms2 and Msh2 genotypes were determined by PCR (14,16), as were the genotypes of the lacI (25) and supF transgenes (23). Three mice (two males and one female) nullizygous for the Pms2 gene and carrying the lacI gene, all 34 weeks old, were chosen for analysis. Previous data generated from Msh2+/+/lacI and Msh2//lacI mice were used for comparison (22). Three Msh2+//supF mice and six Msh2+/+/supF mice, all 6 weeks of age, were also analyzed and compared with data previously generated from Pms2+/+/supF and Pms2//supF mice (23).
Determination of mutation frequency and spectrum with the various reporter genes
DNA was isolated from tissues from lacI transgenic mice as previously described (25). The lacI phage transgenes were excised from chromosomal DNA and packaged into phage particles with Transpack phage packaging extract (Stratagene). Phage containing rescued lacI transgenes were plated on SCS-8 bacterial lawns (Stratagene) in the presence of X-gal. Mutants appeared as blue plaques among the non-mutant, colorless plaques. Mutants were verified by replating and sequenced using various primers spanning the lacI gene (25).
For the supF reporter gene assay, DNA was isolated and packaged and plated as before (23). Functional supF genes suppress the nonsense mutation in the host bacteria ß-galactosidase gene, yielding blue plaques, whereas mutations inactivating the supF gene produce colorless phage plaques. Mutants were sequenced as previously described (23).
The cII gene of phage
can also be used as a reporter gene in
-based transgenic mice, offering the advantage of assessing a different mutational target sequence (28). The cII assay was performed according to the method of Jakubczak et al. (28).
For all reporter gene assays, mutation frequency was obtained by counting a minimum of 100 000 p.f.u./tissue/animal to provide a reliable ratio of mutants to non-mutants.
Statistical comparisons
Raw numbers of mutants observed, over total number of plaques screened, were used for statistical comparisons of mutation frequencies. Pairwise comparisons between mutation frequencies from Msh2/ and Pms2/ were performed using a
2 test of homogeneity (df = 1) for each reporter gene used and data for Msh2/ from all three reporter genes were compared with Pms2/ data, also using a
2 test of homogeneity (df = 2).
Sequence spectra were also analyzed using a
2 test of homogeneity.
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Results
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Higher spontaneous mutation frequencies in Msh2/ versus Pms2/ mice
Mutation frequencies were determined for DNA from several mouse tissues using lacI, supF and cII as target genes and compared with wild-type mice (Figure 1
; 22,23). The supF and cII data are not independent, as the assays are done with linked phage
reporters. With the lacI and supF reporter assays, mutation frequencies for Msh2+/ or Pms2+/ heterozygous mice were not significantly different from those of wild-type mice. However, mutation frequencies in lacI from Msh2 and Pms2 nullizygous mice were 15- and 10-fold higher, respectively, than those of wild-type mice. When the supF gene was used, Msh2/ mice had a 163-fold increase in mutation frequency over controls and Pms2/ mice had, on average, a 100-fold increase (23). Using the cII gene to determine mutation frequencies, Msh2 and Pms2 null mice were 45- and 5-fold higher than wild-type mice, respectively. Interestingly, Msh2-deficient mice had consistently higher mutation frequencies than Pms2-deficient mice with all assay systems when analyzed by
2 test of homogeneity (all P < 0.0000, df = 1) (Figure 1
).

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Fig. 1. Mutation frequencies determined using either the lacI, supF or cII reporter gene are shown for wild-type mice and mice deficient in either Msh2 or Pms2.
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Pms2/ mutation spectrum differs from that of Msh2/ mice
LacI, supF and cII mutants were isolated, purified and sequenced. Mutations isolated more than once per tissue per animal were included only once in the mutation frequency and spectrum data to eliminate any potential bias arising from clonal expansion of a particular mutation. However, this was impossible with the +/1 bp insertions/deletions at particular non-random target sequences for mutation in the supF and cII genes. Thus, the sequenced mutants are all independent, with the exception of the ambiguous +/1 bp frameshift mutations.
When lacI mutants collected from Pms2/ thymus and small intestine were sequenced, transitions were the predominant mutation (58% of all mutations), followed by 1 bp deletions (28%), transversions (8%) and +1 bp insertions (6%) (Table I
). The lacI mutations from Msh2/ mutants were 64% transitions, 21% 1 bp deletions, 13% transversions and 2% +1 bp insertions, which was significantly different from the breakdown of mutations from the Pms2/ mice when compared by
2 analysis (P < 0.001, df = 3).
When supF mutants from Msh2/ and Pms2/ mice were analyzed, the spectra were very different from those obtained using the lacI reporter (Table I
). This is consistent with previous findings that the supF gene is highly sensitive to +/1 bp insertion and deletion mutations occurring within coding runs of C7 and G8 mononucleotide repeats. However, in comparing Msh2- and Pms2-deficient animals, deletions were five times more prevalent than insertions in Msh2 nulls, whereas mutants recovered from Pms2-deficient mice included relatively fewer deletions and more insertions, at a ratio of 2:1 (Table I
;
2 analysis, P > 0.0001, df = 1).
The cII gene provided a third reporter gene sequence for mutational comparisons. Msh2/ mice demonstrated transversions and transitions as the predominant mutation type, with 1 bp deletions again outweighing +1 bp insertions (Table I
). Pms2/ mice demonstrated mostly frameshift mutations, with no transversions; a similar mutation spectrum to that seen with the lacI and supF target genes. The breakdowns of mutation types in Msh2/ and Pms2/ mutants were significantly different by
2 test of homogeneity (P < 0.01, df = 3). Pms2/ mice had proportionately twice as many +1 bp insertions as Msh2/ mice, the ratio being consistent with that determined using the other two reporter genes.
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Discussion
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The model of MMR suggests that both Msh2 and Pms2 are essential for MMR and the absence of either one would have similar consequences with respect to mutation accumulation. Our previous studies with different reporter genes suggested, however, that greater mutability might result from Pms2 deficiency (22,23). The Pms2//supF mice, containing the supF target gene with two mononucleotide runs of C7 and G8, demonstrated a mutation frequency that was 100-fold higher than that of controls (23). In contrast, Msh2/ mice had a mutation frequency only 13-fold higher than background as determined by the lacI transgene, a target, however, that does not contain a mononucleotide run greater than five bases (A5 at position 135). In order to directly compare the effects of Pms2 or Msh2 hemizygosity and nullizygosity on spontaneous mutation, in this study Pms2/ and Msh2/ mice were crossed with three reporter genes for direct comparison of mutation frequency and spectra.
In this more comprehensive analysis, Msh2/ mice consistently demonstrated higher mutation frequencies than the Pms2/ animals (P < 0.001, by
2 test of homogeneity) (Figure 1
). Although there is some variability in that the differences in mutation frequencies ranged from 1.5-fold higher with the lacI gene to 7-fold higher with the cII gene, the results suggest that there is a measurable difference in vivo in the degree of genetic instability caused by Msh2 versus Pms2 deficiency.
The comparison between Pms2//lacI and Pms2//supF mice demonstrates the importance of the target gene when determining mutation frequencies or rates, as mutation frequencies were ~6-fold lower with lacI than with the supF transgene in Pms2/ mice (23). The increased number of mutations in mononucleotide repeats in Pms2//supF compared with Pms2//lacI confirms that genes containing such repeats are at high risk for mutation in MMR deficiency. This study therefore also highlights the importance of the particular reporter gene used when assessing mutation patterns. For example, the lacI gene is most useful for the detection of base substitutions, whereas the supF gene is particularly well suited to detection of frameshift mutations occurring at mononucleotide repeats. The cII gene detects both point mutations as well as frameshifts, as it contains two coding N6 mononucleotide runs.
Pms2//lacI mutants demonstrated a higher proportion of frameshift mutations (34 versus 23%) and a lower proportion of transversions (8 versus 13%) than that observed in Msh2-deficient mice using the same reporter, suggesting that these two mismatch repair deficiencies may not be equivalent in terms of error correction. An increase in the proportion of slippage mutations in Pms2//lacI animals at mononucleotide and dinucleotide repeats, consistent with mutations resulting from polymerase errors, suggests that Pms2 has a major role in the pathway correcting slippage errors and that it has a lesser role in correcting base substitution mismatches, in which case other MutL homologs may be sufficient.
A recent manuscript by Yao et al. (29) also supports the findings that the MMR proteins are not equivalent with respect to the resulting mutator phenotypes in their absence (29). By assessing microsatellite instability and mutation frequency using the supF gene in Mlh1/ and Pms2/ mice, Yao et al. demonstrated that Mlh1/ mice had a higher mutation frequency (1.5- to 3.5-fold) than Pms2/ mice. Furthermore, the increase in mutations at mononucleotide repeats in Mlh1 null mice could be accounted for by an increase in the number of contraction mutations.
The results presented in this paper, as well as the findings of Yao et al. (29), suggest that there could be quantitative as well as qualitative differences in spontaneous mutations depending on which component of the mismatch repair complex is absent. As with Mlh1 null mice, the Msh2 nulls show a relative increase in 1 bp deletions and a slight decrease in +1 bp insertions when compared with the Pms2 null animals. Hence, the combined data suggest that different types of frameshift intermediates may be recognized and/or repaired in different manners. The differences with respect to mutation phenotype may be indicative of differential repair of polymerase slippage events depending on whether they occurred on the leading or lagging strand. If expansion mutations are most likely due to slippage of the nascent strand and deletion mutations result primarily from slippages on the template strand (30), the MMR complex may be associated with the replication machinery in such a way that particular subsets of MMR proteins recognize or act on particular pre-mutational events. For example, Pms2 might be positioned within a post-replication complex such that it is primarily involved in the recognition or repair of nascent strand slippages, whereas Msh2 and Mlh1 may have roles in the monitoring and correction of both template and nascent strand slippage errors. This model would be consistent with the finding of relatively more deletion mutations in the absence of Msh2 and Mlh1 as opposed to Pms2.
Our data suggesting that MMR proteins have differing roles in error correction is in keeping with mutation spectra derived from comparisons performed in cell lines lacking either MSH2 or MLH1, suggesting that each gene defect causes a specific mutator phenotype (31). Also, analysis of immunoglobin gene variable region hypermutation in Msh2- and Pms2-deficient mice revealed different patterns of mutation depending on which gene was absent, suggesting non-equivalent roles for these two proteins in this process (32).
The differences in mutation frequency and spectra obtained from the reporter genes used in this study suggest that cells from patients having germline mutations in MSH2 or PMS2 may be prone to both quantitative and qualitiative differences in genomic instability once the wild-type allele is inactivated. Thus, the growth control genes at risk for mutation may differ depending on the particular MMR gene lost. Pms2/ and Msh2/ mice differ considerably with respect to the incidence of small intestinal neoplasia (13,17). However, the prevalence of PMS2+/ mutations in HNPCC kindreds is too low to allow conclusions about PMS2/-deficient tumor predisposition in affected individuals. The lower incidence of Pms2 germline mutations detected in HNPCC patients (24) may be explained in part, however, by the differences in MMR protein function reported here. The effect of Msh2 deficiency on genome instability is more severe than that of Pms2, hence, critical downstream target genes for carcinogenesis may differ in their risk of mutation depending on which MMR function is lost.
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Notes
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4 To whom correspondence should be addressed Email: seandrew{at}gpu.srv.ualberta.ca 
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Acknowledgments
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We thank J.Penney and L.A.Waddleton in the Transgenic Unit of the Centre for Molecular Medicine and Therapeutics and Keith Ficter at the Canadian Human Genetic Diseases Network Sequencing Core Facility. We also thank Drs A.Buermeyer and S.Baker for stimulating discussions related to this study. This work was supported by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society (to F.R.J.), and by the USPHS (ES05775 to P.M.G.). S.E.A. held a Medical Research Council of Canada Post-Doctoral Fellowship award. A.B.F. held a Natural Sciences and Engineering Research Council of Canada Post-Graduate Scholarship award.
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Received October 1, 1999;
revised March 27, 2000;
accepted March 29, 2000.