Abstract

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII , in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6 . We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1 -deficient mice and Msh2 -deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

Introduction

The DNA mismatch repair (MMR) system in humans is essential in order to maintain genomic stability. MMR deficiency has been shown to result in an increased risk of developing cancer, particularly hereditary non-polyposis colorectal cancer (HNPCC). Cells deficient in MMR exhibit genetic instability manifested as microsatellite instability (MSI) and point mutations. MSI, and thus the loss of MMR, has been seen in a variety of sporadic as well as familial cancers, including endometrial, lung, breast, pancreatic, gastric and prostate ( 1 ). The MMR system functions to preserve genomic stability not only by base mispair correction but also by stimulating apoptosis in response to DNA damage caused by physical and chemical agents ( 2 ).

In mammalian cells there are multiple homologs of the Escherichia coli MutS and MutL proteins. The MutS homologs, MSH2, MSH3 and MSH6, are crucial for mismatch recognition. MSH2 forms heterodimers with either MSH6 (MutSα) or MSH3 (MutSβ). The MutL homologs include MLH1, PMS1 and PMS2. Both of the MutS complexes interact with the MutLα heterodimer, which consists of MLH1 and PMS2. The MutLα complex appears to play the key role in the process of mismatch correction by linking the mismatch recognition of MutSα and MutSβ with the excision of mutations and resynthesis of corrected bases ( 3 ). More recently, the novel MMR gene Mlh3 has been shown to interact with Mlh1 and contribute to MMR as well ( 4–6 ). In addition, a number of homologs, such as MSH4 and MSH5, have been identified that have not been linked to hereditary cancer, and may participate in meiosis and other cellular functions besides mismatch recognition ( 7 ). MLH3 has also been shown to play a crucial role in mammalian meiosis ( 8 ).

Since there is such a variety of MutS and MutL homologs in mammalian cells, it has been proposed that there is both divergence as well as redundancy of function. For example, the MutSα complex recognizes single base pair point mutations and small insertion/deletion loops (IDLs) and the MutSβ complex recognizes base pair mismatches and large IDLs ( 3 , 7 ).

In the human syndrome of HNPCC, affected individuals primarily have mutations in MSH2 or MLH1 , but defects in MSH6, PMS1 and PMS2 , although less common, have also been shown to be a factor ( 3 , 9–11 ). Many sporadic cancers have also been found to display MMR deficiency due to the silencing of the MLH1 gene via promoter hypermethylation ( 10 ).

A number of studies have examined genetic instability in the setting of MMR deficiency using both cell culture and mouse models ( 6 , 12–17 ). The initial mouse studies have begun to define in vivo patterns of mutations in the presence of MMR deficiency ( 6 , 15–17 ). However, most previous studies have focused on individual genotypes or on pairwise comparisons. To more systematically compare the functional roles of the MutS and MutL homologs in mammalian cells, we utilized a series of knock-out mouse models to examine patterns of genetic instability in vivo due to a deficiency in Pms2, Mlh1, Msh2, Msh3, Msh6, Msh2 and Msh3 , or Msh3 and Msh6 . On the basis of the studies mentioned above, as well as the fact that MSH2 and MLH1 are the genes most commonly associated with HNPCC-affected individuals, we hypothesized that the Mlh1 −/− and Msh2 −/− mice would have the highest mutation frequencies. We also hypothesized that the Pms2 −/− , Msh6 −/− and Msh3 −/− mice would have intermediate levels of mutations, with the relative pattern potentially providing insight into the association of these genotypes with cancer predisposition.

This work was facilitated by the use of transgenic mice carrying either the supFG1 or the cII mutation reporter genes within chromosomally integrated, recoverable lambda shuttle vectors. These reporter genes and lambda vector constructs serve as powerful tools for directly studying mutagenesis in vivo and their relatively short lengths, sensitivity and low background of spontaneous mutation frequencies allow accurate determination of the frequencies or types of mutations due to MMR deficiency.

We found that the extent to which genetic instability, measured by the frequencies of mutations in two different reporter transgenes, is affected in mice deficient in the MMR genes varied considerably, but that all the groups of MMR-deficient mice had mean mutation frequencies significantly higher than the mean mutation frequencies of wild-type mice. Among the MMR single nullizygous mice, Mlh1 and Msh2 deficiency produced the greatest instability, whereas Msh3 deficiency generated the least. Compared with wild-type, the double mutant mice deficient for both Msh2 and Msh3 or deficient for both Msh3 and Msh6 displayed the largest increases in mutation frequencies of all the groups. In addition, examination of the mutation patterns indicated differences that may provide insight into the role of the various MMR proteins in mismatch correction. The results presented here also add to the increasing amount of evidence that the MMR factors play both distinctive and redundant roles in DNA repair.

Materials and methods

Transgenic mice and genotyping

Mice nullizygous for the Pms2 ( 18 ), Mlh1 ( 19 ), Msh2 ( 20 ), Msh3 ( 21 ) or Msh6 ( 22 ) genes were crossed with transgenic mouse lines carrying either the supFG1 (3340 strain) or cII (Muta™ Mouse strain, Covance of Denver, PA, USA) mutation reporter genes within chromosomally integrated, lambda shuttle vectors to create MMR +/+ /supFG1, MMR +/− /supFG1, MMR −/− /supFG1, MMR +/+ /cII, MMR +/− /cII and MMR −/− /cII offspring. All mice were derived from the C57BL/6 mouse background, and construction of the supFG1 and cII lambda shuttle vectors has previously been described ( 13 , 23 ). Offspring that contained either the supFG1 or cII mutational reporter genes and heterozygous for each of the above-listed MMR genes were bred to create sibling sets of wild-type and nullizygous mice to be studied for mutational analysis. Heterozygotes were used for breeding to continue to create sibling sets of wild-type and nullizygous mice. All genotypes were determined by polymerase chain reaction (PCR) of tail DNA as described previously for Pms2 ( 18 ), Mlh1 ( 19 ), Msh2 ( 20 ), Msh3 ( 21 ) and Msh6 ( 21 ). The presence or absence of the supFG1 or cII reporter gene was also determined by PCR as described previously ( 13 , 23 , 24 ).

For both the supFG1 and cII lines, Msh2 +/− mice were bred with Msh3 +/− in order to generate mice heterozygous for both MMR genes. These double heterozygotes were then bred to generate mice deficient for both Msh2 and Msh3 . The same procedure was followed to produce mice deficient for both Msh2 and Msh6 and for both Msh3 and Msh6 . However, we were unable to obtain mice deficient for both Msh2 and Msh6 in spite of 37 breeding attempts and analysis of 218 pups. Genotypes were determined as described above.

λ Shuttle vector rescue and mutagenesis assays

Sibling mice either wild-type or nullizygous for each of the five MMR genes and containing either the supFG1 or cII reporter genes were killed at age 10–12 weeks by cervical dislocation. Skin (epidermis plus superficial dermis) and proximal colon (smooth muscle layers plus epithelium) samples were collected, quick frozen in liquid nitrogen and stored at −80°C. A total of 2–4 mice were killed for each of the 10 genotypes.

The procedure described above was also performed with Msh2 −/− /Msh3 −/− mice and Msh3 −/− /Msh6 −/− mice containing either the supFG1 or cII reporter genes. A total of 2–3 mice were killed for each of these four genotypes.

High molecular weight DNA was prepared from the skin and colon samples as described previously ( 24 ). We chose to use the skin and colon because previous work in our laboratory has shown these tissues to provide good DNA preparation for efficient λ in vitro packaging. λ in vitro packaging extracts were made as described previously and were used to rescue phage vectors from skin and colon DNA ( 13 , 24–27 ). Rescued phage were analyzed for supFG1 and cII mutations as described previously ( 13 , 24 ). Briefly, functional supFG1 reporter genes suppress the nonsense mutation in the host bacteria β-galactose gene, yielding blue plaques, whereas mutations inactivating the supFG1 gene produce colorless phage plaques ( 13 ). The plates were screened for colorless plaques and the total number of plaques per plate was calculated. For the cII reporter gene, at 24°C wild-type phage form lysogens and therefore do not form plaques. However, phage with inactivating mutations can be detected through lytic plaque production after 48 h at this temperature ( 28 ). The mutants produced at 24°C were counted and the total number of packaged phage was determined by titers plated at 37°C, which allows lytic plaque production regardless of cII status ( 28 ).

Mutant sequencing

A portion of mutant plaques from each of the 14 groups of knock-out mice were picked immediately upon detection and plaque-purified. The plaque-purified mutants were amplified by PCR and purified by the Qiagen PCR purification kit. Sequence analysis was performed as described ( 13 ). Primers used to amplify the supFG1 or cII region from the DNA and for sequencing have been described previously ( 13 , 23 ).

Statistics

Mutation frequencies were defined as the number of mutant plaques per the total number of plaque forming units from skin and colon DNA of each mouse. The raw data of the wild-type mice (separately for each of the two reporter genes) were combined and the means of the skin and colon mutation frequencies were used to compare the MMR nullizygous mice. Statistical differences among mutation frequencies were determined by the unpaired t -test (Prism Version 4, GraphPad Software). The mutation frequency for each group of mice is expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined as P < 0.05.

Results

Generation of mice deficient in selected MMR factors

Mice were bred as described in Materials and methods in an attempt to generate a series of transgenic mice containing either the supFG1 or cII mutation reporter gene (in a chromosomally recoverable lambda phage vector) along with specific genotypes at MMR loci as follows: (i) WT ; (ii) Pms2 null; (iii) Mlh1 null; (iv) Msh2 null; (v) Msh3 null; (vi) Msh6 null; (vii) Msh2 and Msh3 double null; (viii) Msh2 and Msh6 double null; and (ix) Msh3 and Msh6 double null. All desired genotypes were obtained except for Msh2 and Msh6 double nulls, in spite of numerous attempts.

Mutation frequencies in supFG1 mice

The mean mutation frequencies were determined for the skin and colon DNA of the supFG1 /wild-type, supFG1/Pms2 −/− , supFG1/Mlh1 −/− , supFG1/Msh2 −/− , supFG1/Msh3 −/− , supFG1/Msh6 −/− , supFG1/Msh2 −/− /Msh3 −/− and supFG1/Msh3 −/− /Msh6 −/− mice. The skin and colon sub-totals for the mutants/plaques were combined since there were no significant differences found between the two tissues (data not shown). Table I conveys the total number of mutants per the total number of plaques counted for each of the 12 groups of mice, including wild-type (Supplementary Table IV presents the mutants/plaques and mutation frequencies for each individual animal), and Figure 1 presents a graphical comparison of the mean mutation frequencies. The overall average frequency of mutations in the skin and colon DNA of the wild-type supFG1 mice ( Figure 1 ) is consistent with baseline mutation frequencies previously observed in such transgenic animal systems ( 13 , 28 ).

Fig. 1

Comparison of supFG1 reporter gene mutation frequencies in wild-type and MMR-deficient mice. The mutation frequency for each group of mice is expressed as the mean ± SEM. The wild-type mutation frequency is the mean ± SEM of the skin and colon mutation frequencies of all the wild-type mice listed in Table I . The absolute frequencies of the types of mutations are indicated by the shaded segments of each bar. A total of 74 mutants were analyzed for the Pms2 −/− mice, 81 for the Mlh1 −/− mice, 59 for the Msh2 −/− mice, 64 for the Msh3 −/− mice, 62 for the Msh6 −/− mice, 24 for the Msh2 / /Msh3 / mice and 24 for the Msh3 / /Msh6 / mice.

Table I

Mutation frequencies in wild-type and MMR-deficient mice in the supFG1 and cII reporter genes

MMR gene deficiency Number of mice Wild-type Nullizygous


Mutants/total plaques Frequency (× 10 −5 ) a Mutants/total plaques Frequency (×10 −5 ) a
supFG1
     Pms2 3 WT, 3 KO 32/381 363 9.2 664/277 756 220.7
     Mlh1 3 WT, 4 KO 59/525 640 11.3 4560/635 452 616.8
     Msh2 2 WT, 3 KO 35/331 299 10.4 2820/467 502 555.1
     Msh3 3 WT, 3 KO 55/680 415 8.2 84/557 240 14.5
     Msh6 2 WT, 3 KO 8/210 291 3.8 469/409 987 116.0
     Msh2/Msh3 3 KO/KO 4065/507 039 777.8
     Msh3/Msh6 3 KO/KO 4419/490 062 873.4
cII
     Pms2 2 WT, 3 KO 18/530 354 3.4 186/788 314 23.6
     Mlh1 3 WT, 4 KO 122/1 952 754 6.4 381/1 163 005 34.5
     Msh2 4 WT, 3 KO 53/1 106 760 6.1 329/904 323 35.7
     Msh3 4 WT, 3 KO 56/1 121 690 5.0 78/784 595 9.7
     Msh6 3 WT, 2 KO 51/1 103 242 4.6 33/395 894 9.4
     Msh2/Msh3 2 KO/KO 849/1 296 294 53.9
     Msh3/Msh6 3 KO/KO 1399/1 465 170 90.5
MMR gene deficiency Number of mice Wild-type Nullizygous


Mutants/total plaques Frequency (× 10 −5 ) a Mutants/total plaques Frequency (×10 −5 ) a
supFG1
     Pms2 3 WT, 3 KO 32/381 363 9.2 664/277 756 220.7
     Mlh1 3 WT, 4 KO 59/525 640 11.3 4560/635 452 616.8
     Msh2 2 WT, 3 KO 35/331 299 10.4 2820/467 502 555.1
     Msh3 3 WT, 3 KO 55/680 415 8.2 84/557 240 14.5
     Msh6 2 WT, 3 KO 8/210 291 3.8 469/409 987 116.0
     Msh2/Msh3 3 KO/KO 4065/507 039 777.8
     Msh3/Msh6 3 KO/KO 4419/490 062 873.4
cII
     Pms2 2 WT, 3 KO 18/530 354 3.4 186/788 314 23.6
     Mlh1 3 WT, 4 KO 122/1 952 754 6.4 381/1 163 005 34.5
     Msh2 4 WT, 3 KO 53/1 106 760 6.1 329/904 323 35.7
     Msh3 4 WT, 3 KO 56/1 121 690 5.0 78/784 595 9.7
     Msh6 3 WT, 2 KO 51/1 103 242 4.6 33/395 894 9.4
     Msh2/Msh3 2 KO/KO 849/1 296 294 53.9
     Msh3/Msh6 3 KO/KO 1399/1 465 170 90.5

a Mutation frequency was determined as the average of the means.

Table I

Mutation frequencies in wild-type and MMR-deficient mice in the supFG1 and cII reporter genes

MMR gene deficiency Number of mice Wild-type Nullizygous


Mutants/total plaques Frequency (× 10 −5 ) a Mutants/total plaques Frequency (×10 −5 ) a
supFG1
     Pms2 3 WT, 3 KO 32/381 363 9.2 664/277 756 220.7
     Mlh1 3 WT, 4 KO 59/525 640 11.3 4560/635 452 616.8
     Msh2 2 WT, 3 KO 35/331 299 10.4 2820/467 502 555.1
     Msh3 3 WT, 3 KO 55/680 415 8.2 84/557 240 14.5
     Msh6 2 WT, 3 KO 8/210 291 3.8 469/409 987 116.0
     Msh2/Msh3 3 KO/KO 4065/507 039 777.8
     Msh3/Msh6 3 KO/KO 4419/490 062 873.4
cII
     Pms2 2 WT, 3 KO 18/530 354 3.4 186/788 314 23.6
     Mlh1 3 WT, 4 KO 122/1 952 754 6.4 381/1 163 005 34.5
     Msh2 4 WT, 3 KO 53/1 106 760 6.1 329/904 323 35.7
     Msh3 4 WT, 3 KO 56/1 121 690 5.0 78/784 595 9.7
     Msh6 3 WT, 2 KO 51/1 103 242 4.6 33/395 894 9.4
     Msh2/Msh3 2 KO/KO 849/1 296 294 53.9
     Msh3/Msh6 3 KO/KO 1399/1 465 170 90.5
MMR gene deficiency Number of mice Wild-type Nullizygous


Mutants/total plaques Frequency (× 10 −5 ) a Mutants/total plaques Frequency (×10 −5 ) a
supFG1
     Pms2 3 WT, 3 KO 32/381 363 9.2 664/277 756 220.7
     Mlh1 3 WT, 4 KO 59/525 640 11.3 4560/635 452 616.8
     Msh2 2 WT, 3 KO 35/331 299 10.4 2820/467 502 555.1
     Msh3 3 WT, 3 KO 55/680 415 8.2 84/557 240 14.5
     Msh6 2 WT, 3 KO 8/210 291 3.8 469/409 987 116.0
     Msh2/Msh3 3 KO/KO 4065/507 039 777.8
     Msh3/Msh6 3 KO/KO 4419/490 062 873.4
cII
     Pms2 2 WT, 3 KO 18/530 354 3.4 186/788 314 23.6
     Mlh1 3 WT, 4 KO 122/1 952 754 6.4 381/1 163 005 34.5
     Msh2 4 WT, 3 KO 53/1 106 760 6.1 329/904 323 35.7
     Msh3 4 WT, 3 KO 56/1 121 690 5.0 78/784 595 9.7
     Msh6 3 WT, 2 KO 51/1 103 242 4.6 33/395 894 9.4
     Msh2/Msh3 2 KO/KO 849/1 296 294 53.9
     Msh3/Msh6 3 KO/KO 1399/1 465 170 90.5

a Mutation frequency was determined as the average of the means.

With respect to the MMR-deficient mice, all the groups exhibited significantly higher mutation frequencies than the wild-type mice (pairwise statistical comparisons between each genotype are shown in Table II ). Mlh1 -deficient mice exhibited the highest mutation frequencies among the supFG1 single nullizygous mice, >72 times the mutation frequencies of wild-type mice ( Figure 1 ). The mutation frequencies of the Msh2 -deficient mice were also quite elevated, with a 65-fold increase above wild-type ( Figure 1 ). Of the supFG1 /MMR-deficient mice, the Msh3 -deficient mice exhibited the lowest, yet still significantly elevated, mutation frequencies ( Table II and Figure 1 ).

Table II

Pairwise tests of statistical significance in comparisons of supFG1 and cII reporter gene mutation frequencies in wild-type and MMR-deficient mice

Msh3/Msh6 Msh2/Msh3 Msh6 Msh3 Msh2 Mlh1 Pms2
supFG1
     WT <0.001 <0.001 <0.001 0.044 <0.001 <0.001 <0.001
     Pms2 0.007 0.004 0.038 <0.001 0.014 0.009
     Mlh1 0.205 0.344 0.001 <0.001 0.675
     Msh2 0.134 0.201 0.002 <0.001
     Msh3 <0.001 <0.001 0.017
     Msh6 <0.001 <0.001
     Msh2/Msh3 0.663
cII
     WT <0.001 <0.001 0.031 0.027 <0.001 <0.001 <0.001
     Pms2 0.0011 0.0322 0.0397 0.0341 0.3321 0.2165
     Mlh1 0.0056 0.2070 0.0270 0.0174 0.6840
     Msh2 0.0028 0.1067 0.0205 0.0139
     Msh3 0.0006 0.0080 0.8512
     Msh6 0.0017 0.0159
     Msh2/Msh3 0.1101
Msh3/Msh6 Msh2/Msh3 Msh6 Msh3 Msh2 Mlh1 Pms2
supFG1
     WT <0.001 <0.001 <0.001 0.044 <0.001 <0.001 <0.001
     Pms2 0.007 0.004 0.038 <0.001 0.014 0.009
     Mlh1 0.205 0.344 0.001 <0.001 0.675
     Msh2 0.134 0.201 0.002 <0.001
     Msh3 <0.001 <0.001 0.017
     Msh6 <0.001 <0.001
     Msh2/Msh3 0.663
cII
     WT <0.001 <0.001 0.031 0.027 <0.001 <0.001 <0.001
     Pms2 0.0011 0.0322 0.0397 0.0341 0.3321 0.2165
     Mlh1 0.0056 0.2070 0.0270 0.0174 0.6840
     Msh2 0.0028 0.1067 0.0205 0.0139
     Msh3 0.0006 0.0080 0.8512
     Msh6 0.0017 0.0159
     Msh2/Msh3 0.1101

Probability values were obtained with the unpaired t -test.

Statistically significant results are in bold ( P < 0.05).

Table II

Pairwise tests of statistical significance in comparisons of supFG1 and cII reporter gene mutation frequencies in wild-type and MMR-deficient mice

Msh3/Msh6 Msh2/Msh3 Msh6 Msh3 Msh2 Mlh1 Pms2
supFG1
     WT <0.001 <0.001 <0.001 0.044 <0.001 <0.001 <0.001
     Pms2 0.007 0.004 0.038 <0.001 0.014 0.009
     Mlh1 0.205 0.344 0.001 <0.001 0.675
     Msh2 0.134 0.201 0.002 <0.001
     Msh3 <0.001 <0.001 0.017
     Msh6 <0.001 <0.001
     Msh2/Msh3 0.663
cII
     WT <0.001 <0.001 0.031 0.027 <0.001 <0.001 <0.001
     Pms2 0.0011 0.0322 0.0397 0.0341 0.3321 0.2165
     Mlh1 0.0056 0.2070 0.0270 0.0174 0.6840
     Msh2 0.0028 0.1067 0.0205 0.0139
     Msh3 0.0006 0.0080 0.8512
     Msh6 0.0017 0.0159
     Msh2/Msh3 0.1101
Msh3/Msh6 Msh2/Msh3 Msh6 Msh3 Msh2 Mlh1 Pms2
supFG1
     WT <0.001 <0.001 <0.001 0.044 <0.001 <0.001 <0.001
     Pms2 0.007 0.004 0.038 <0.001 0.014 0.009
     Mlh1 0.205 0.344 0.001 <0.001 0.675
     Msh2 0.134 0.201 0.002 <0.001
     Msh3 <0.001 <0.001 0.017
     Msh6 <0.001 <0.001
     Msh2/Msh3 0.663
cII
     WT <0.001 <0.001 0.031 0.027 <0.001 <0.001 <0.001
     Pms2 0.0011 0.0322 0.0397 0.0341 0.3321 0.2165
     Mlh1 0.0056 0.2070 0.0270 0.0174 0.6840
     Msh2 0.0028 0.1067 0.0205 0.0139
     Msh3 0.0006 0.0080 0.8512
     Msh6 0.0017 0.0159
     Msh2/Msh3 0.1101

Probability values were obtained with the unpaired t -test.

Statistically significant results are in bold ( P < 0.05).

The mutation frequencies observed in the Msh2 −/− /Msh3 −/− double knock-out mice were significantly greater than those of the wild-type mice (by over 90-fold), as well as those of the Pms2 −/− , Msh6 −/− and Msh3 −/− mice ( Table II and Figure 1 ). The mutation frequencies of the Msh3 −/− /Msh6 −/− double knock-out mice were >100-fold higher than the wild-type mutation frequencies and also significantly higher than those of the Pms2 −/− , Msh6 −/− and Msh3 −/− mice ( Table II and Figure 1 ).

Mutation frequencies in cII mice

The mean mutation frequencies were determined for the skin and colon DNA of the cII /wild-type, cII/Pms2 −/− , cII/Mlh1 −/− , cII/Msh2 −/− , cII/Msh3 −/− , cII/Msh6 −/− , cII/Msh2 −/− /Msh3 −/− and cII/Msh3 −/− /Msh6 −/− mice. Similar to the supFG1 mice, the skin and colon mutants/plaques were combined since there were no significant differences found between the two tissues (data not shown). Table I presents the total number of mutants per the total number of plaques counted for each of the mouse groups (Supplementary Table V displays the sub-totals of mutants/plaques and mutation frequencies for each individual animal) and Figure 2 presents a graphical comparison of the mean mutation frequencies. The overall average of the skin and colon DNA mutation frequencies of the wild-type cII mice ( Figure 2 ) is, as in the case of the supFG1 gene, consistent with previous studies of baseline cII mutation frequencies in other studies of lambda vector-containing transgenic mice ( 28 ).

Fig. 2

Comparison of cII reporter gene mutation frequencies in wild-type and MMR-deficient mice. The mutation frequency for each group of mice is expressed as the mean ± SEM. The wild-type mutation frequency is the mean ± SEM of the skin and colon mutation frequencies of all the wild-type mice listed in Table I . The absolute frequencies of the types of mutations are indicated by the shaded segments of each bar. A total of 27 mutants were analyzed for the Pms2 −/− mice, 33 for the Mlh1 −/− mice, 31 for the Msh2 −/− mice, 28 for the Msh3 −/− mice, 15 for the Msh6 −/− mice, 23 for the Msh2 −/− /Msh3 −/− mice and 22 for the Msh3 −/− /Msh6 −/− mice.

On the basis of the pairwise statistical comparisons shown in Table II , all of the groups of cII mice exhibited significantly higher mutation frequencies than the wild-type mice. The Msh2 -deficient mice and the Mlh1 -deficient mice exhibited the highest mutation frequencies among the single nullizygous mice ( Figure 2 ), with both having mutation frequencies >7-fold greater than the wild-type mice. The Msh3 -deficient and Msh6 -deficient mice presented the lowest mutation frequencies of the cII mice, but these were still significantly higher than those of the wild-type mice ( Table II and Figure 2 ).

The mutation frequencies seen in the double nullizygous Msh2 −/− /Msh3 −/− mice were significantly greater than the mutation frequencies of the wild-type, Pms2 −/− , Msh6 −/− and Msh3 −/− mice ( Table II ). Interestingly, the mutation frequencies of the Msh3 −/− /Msh6 −/− mice were significantly higher than all of the single nullizygous groups of mice ( Table II and Figure 2 ).

Sequence analysis of supFG1 and cII mutants

A sample of the mutants from each of the MMR-deficient mice, for both the reporter genes, was analyzed by DNA sequencing, and the patterns of mutations were determined. No differences were detected between the mutants of skin and colon for either of the reporter genes (data not shown). In the case of each genotype, the absolute frequencies of deletions/insertions and transitions/transversions were calculated for each group by multiplying the percentages of each type by their respective mutation frequencies ( Figures 1 and 2 ).

The majority of the supFG1 mutations were either −1 deletion or +1 insertion mutations, with the majority being deletions ( Table III ). There were only slight differences in the types of mutations analyzed among the different genotypes, as only a small proportion of the Msh2 −/− , Pms2 −/− , Msh3 −/− or Msh6 −/− supFG1 mutations consisted of base substitutions; most were deletions or insertions ( Table III and Figure 1 ). This prevalence of deletion and insertion mutations in the supFG1 mice was not surprising due to the two G : C bp stretches (of lengths of 7 and 8 bp) in the supFG1 gene. Those sites are mutation hotspots and account for the majority of the insertion and deletion mutations detected (data not shown).

Table III

Analysis of the mutants sequenced from each group of MMR-deficient mice

Mutation type Pms2 −/− Mlh1 −/− Msh2 −/− Msh3 −/− Msh6 −/− Msh2 −/− /Msh3 −/− Msh3 −/− /Msh6 −/−
supFG1
    Deletions/insertions 71 81 58 57 58 24 24
        −1 nucleotide 47 65 46 39 29 18 14
        +1 nucleotide 24 16 12 18 29 6 10
    Transitions 3 0 1 4 0 0 0
        CG→TA 2 1 4
        TA→CG 1
    Transversions 0 0 0 3 4 0 0
        CG→AT 2 2
        CG→GC 1
        AT→CG 2
cII
    Deletions/insertions 18 18 19 6 2 16 15
        −1 nucleotide 14 16 14 2 2 13 14
        +1 nucleotide 4 2 5 2 0 2
        > −1 nucleotides 2 1 1
    Transitions 4 13 10 17 10 6 7
        CG→TA 2 10 4 12 6 6 6
        TA→CG 2 3 6 5 4 1
    Transversions 5 2 2 5 3 1 0
        CG→AT 2 1 2 1
        CG→GC 3 1 2 2
        AT→TA 2 1 1
Mutation type Pms2 −/− Mlh1 −/− Msh2 −/− Msh3 −/− Msh6 −/− Msh2 −/− /Msh3 −/− Msh3 −/− /Msh6 −/−
supFG1
    Deletions/insertions 71 81 58 57 58 24 24
        −1 nucleotide 47 65 46 39 29 18 14
        +1 nucleotide 24 16 12 18 29 6 10
    Transitions 3 0 1 4 0 0 0
        CG→TA 2 1 4
        TA→CG 1
    Transversions 0 0 0 3 4 0 0
        CG→AT 2 2
        CG→GC 1
        AT→CG 2
cII
    Deletions/insertions 18 18 19 6 2 16 15
        −1 nucleotide 14 16 14 2 2 13 14
        +1 nucleotide 4 2 5 2 0 2
        > −1 nucleotides 2 1 1
    Transitions 4 13 10 17 10 6 7
        CG→TA 2 10 4 12 6 6 6
        TA→CG 2 3 6 5 4 1
    Transversions 5 2 2 5 3 1 0
        CG→AT 2 1 2 1
        CG→GC 3 1 2 2
        AT→TA 2 1 1
Table III

Analysis of the mutants sequenced from each group of MMR-deficient mice

Mutation type Pms2 −/− Mlh1 −/− Msh2 −/− Msh3 −/− Msh6 −/− Msh2 −/− /Msh3 −/− Msh3 −/− /Msh6 −/−
supFG1
    Deletions/insertions 71 81 58 57 58 24 24
        −1 nucleotide 47 65 46 39 29 18 14
        +1 nucleotide 24 16 12 18 29 6 10
    Transitions 3 0 1 4 0 0 0
        CG→TA 2 1 4
        TA→CG 1
    Transversions 0 0 0 3 4 0 0
        CG→AT 2 2
        CG→GC 1
        AT→CG 2
cII
    Deletions/insertions 18 18 19 6 2 16 15
        −1 nucleotide 14 16 14 2 2 13 14
        +1 nucleotide 4 2 5 2 0 2
        > −1 nucleotides 2 1 1
    Transitions 4 13 10 17 10 6 7
        CG→TA 2 10 4 12 6 6 6
        TA→CG 2 3 6 5 4 1
    Transversions 5 2 2 5 3 1 0
        CG→AT 2 1 2 1
        CG→GC 3 1 2 2
        AT→TA 2 1 1
Mutation type Pms2 −/− Mlh1 −/− Msh2 −/− Msh3 −/− Msh6 −/− Msh2 −/− /Msh3 −/− Msh3 −/− /Msh6 −/−
supFG1
    Deletions/insertions 71 81 58 57 58 24 24
        −1 nucleotide 47 65 46 39 29 18 14
        +1 nucleotide 24 16 12 18 29 6 10
    Transitions 3 0 1 4 0 0 0
        CG→TA 2 1 4
        TA→CG 1
    Transversions 0 0 0 3 4 0 0
        CG→AT 2 2
        CG→GC 1
        AT→CG 2
cII
    Deletions/insertions 18 18 19 6 2 16 15
        −1 nucleotide 14 16 14 2 2 13 14
        +1 nucleotide 4 2 5 2 0 2
        > −1 nucleotides 2 1 1
    Transitions 4 13 10 17 10 6 7
        CG→TA 2 10 4 12 6 6 6
        TA→CG 2 3 6 5 4 1
    Transversions 5 2 2 5 3 1 0
        CG→AT 2 1 2 1
        CG→GC 3 1 2 2
        AT→TA 2 1 1

When compared with the supFG1 mutants, the cII mutants showed a more diverse spectrum of mutation types, although there were still larger numbers of deletions and insertions than of base substitutions in the Pms2 −/− , Mlh1 −/− , Msh2 −/− , Msh2 −/− /Msh3 −/− and Msh3 −/− /Msh6 −/− mice ( Table III and Figures 1 and 2 ). However, the majority of the cII mutations in both the Msh3 −/− and Msh6 −/− mice were transitions and transversions ( Table III and Figure 2 ).

Discussion

We have reported here a comprehensive comparison of mutation frequencies and patterns in wild-type and MMR-deficient mice in the absence of genotoxic insults. The spontaneous mutation patterns found in these MMR-deficient mice reveal a hierarchy in the extent to which each of the MMR factors contributes to genome stability, with the highest mutation frequencies among the single nullizygous mice seen in the Mlh1 -deficient and Msh2 -deficient mice, and the least in the Msh3 -deficient mice. These comparisons were performed in the context of two different reporter genes, yielding similar relative values, although the absolute mutation frequencies were different, reflective of the nature of the reporter genes. In this regard, earlier studies have shown that the supFG1 reporter gene is particularly prone to mutagenesis in the absence of MMR owing to two long mononucleotide repeats in the coding sequence ( 28 ). Therefore, it was not surprising that the MMR-deficient supFG1 mice were found to have mutation frequencies that were considerably elevated when compared with the MMR-deficient cII mice. Nonetheless, similar trends were seen in the comparison of MMR genotypes in both the supFG1 and cII reporter lineages.

A number of previous studies have also utilized mutation reporter genes to investigate genome instability in MMR-deficient mice ( 13 , 15–17 , 29–31 ). The data presented here expand upon this prior body of work and provide further validation of these earlier findings. For example, it was previously shown that Msh2 -deficient mice exhibit mutation frequencies higher than Pms2 -deficient mice in the supF, lacI and cII reporter genes ( 15 ). Other studies also have revealed elevated mutation frequencies in Mlh1, Pms2 and Msh6 nullizygous mice, with relative levels consistent with our findings ( 15 , 17 , 29 , 31 ). Additionally, Mlh1 -deficient mice have shown higher supFG1 and lacI mutation frequencies than Pms2 -deficient mice ( 16 , 30 ), and recently MSI analysis showed that Mlh3 −/− /Pms2 −/− mice have higher mutation frequencies than Pms2 −/− mice, but the same as Mlh1 −/− mice ( 6 ). However, the present work also provides a combined analysis of these and additional MMR genotypes, allowing a comprehensive comparison among these MMR factors.

In addition, the work reported here includes an analysis of double nullizygous mice in two different reporter systems. Although Msh3/Msh6 -deficient mice have previously been examined ( 21 , 29 ), this is the first characterization of mice deficient for both Msh2 and Msh3 . The Msh2 −/− /Msh3 −/− and Msh3 −/− /Msh6 −/− mice had the highest mutation frequencies of all the genotypes studied, suggesting that disrupting more than one of the MMR genes escalates the accumulation of mutations, leading to considerably increased mutation frequencies. A similar effect has also been observed in an analysis of hprt gene mutations of colon adenocarcinoma cells with concomitant MLH1 and MSH6 inactivation ( 31 ). Although statistically significant only in the case of the cII Msh3 −/− /Msh6 −/− data and not in the supFG1 data, the increased mutation frequencies that the Msh2 −/− /Msh3 −/− and Msh3 −/− /Msh6 −/− mice displayed over the Msh2 −/− mice raise the possibility that the absence of Msh2 may not completely eliminate MMR in vivo . However, numerous studies support a critical role for Msh2 in mismatch recognition, and there is not yet any biochemical evidence to suggest that there may be an alternative factor. Since MSH3 and MSH6 overlap in function, and MSH2 has been suggested to partition between available pools of the two ( 32 ), the higher frequencies of the Msh3/Msh6 -deficient mice may suggest an increased level of disruption in that there are not other factors to take their place. In any case, the key role of Msh2 in both the MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3) heterodimers is borne out in the comparison among the MutS homologs. In contrast, elimination of either Msh3 or Msh6 alone still maintains some functional MMR activity, consistent with the persistence of the MutSα or MutSβ heterodimers, respectively. Similarly, a non-redundant role for Mlh1 among the MutL homologs is also supported by the data, as presented here and elsewhere ( 6 ).

The greater mutation frequencies of the Msh6 −/− mice compared with Msh3 −/− mice supports the previous conclusion that Msh6 may be more important to the maintenance of genomic stability than is Msh3 . This is also reflected in previous work that has shown that Msh3 deficiency plays a critical role in survival of mice predominantly when Msh6 is also deficient ( 21 ). Also, the loss of Msh6 has been shown to cause a strong predisposition to intestinal tumors in APC 1638N mice, whereas the loss of Msh3 did not ( 33 ). The apparently greater impact of Msh6 deficiency versus Msh3 deficiency may partly explain why our attempts to create Msh2 −/− /Msh6 −/− mice were unsuccessful. However, the simplest explanation is that the close proximity of these loci to each other on mouse chromosome 17 means that they are too closely linked to allow any reasonable chance of the required cross-over events within the scope of the number of progeny evaluated in this work. In any case, in the single nullizygous Msh3 −/− mice we did detect a mutator phenotype. Although lowest among all the MMR-deficient mice studied, the frequencies in the Msh3 −/− mice were still significantly elevated above that of the wild-type mice. In addition, the study mentioned above regarding the comparison of Msh6 versus Msh3 deficiency also reported that Msh3 −/− /Msh6 −/− mice display a cancer predisposition phenotype that is indistinguishable from Mlh1 or Msh2 deficiency ( 21 ). Our results are in keeping with these findings.

Analysis of the types of mutations in the various mice revealed a predominance of deletions in all of the MMR-deficient mice in the context of the supFG1 reporter gene. This reporter gene is most sensitive to +1 or −1 mutations, and so the predominance of −1 mutations suggests a propensity for such replication errors at this locus or a biased role for MMR in fixing products of template slippage leading to −1 deletions. Analysis of the cII mutations revealed a more diverse pattern, but there was still an overall predominance of deletions in five of the seven genotypes examined. Since this gene is well established to report all possible point mutations with less bias than supFG1 , the predominance of deletions in the cII mouse assay suggests that such mutations are the primary in vivo result of MMR deficiency.

Taken together, the results reported here establish a rank order of the severity of MMR deficiency in vivo across five single and two double nullizygous MMR genotypes. All of the MMR genes studied were found to be important in preserving genome stability, with a hierarchy of effect consistent with their proposed single or multiple roles as components of MMR heterodimeric complexes.

Abbreviations

     
  • MMR

    mismatch repair

  •  
  • SEM

    standard error of the mean

We thank L. Cabral, F. Rogers, S. Gibson and P. Hegan for their help. This work was supported by a grant from the USPHS (NIH/NIEHS RO1 ES05775 to P.M.G.).

Conflict of Interest Statement : None declared.

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