Human Molecular Genetics, 2002, Vol. 11, No. 11 1303-1310
© 2002 Oxford University Press
Functional analysis of MSH6 mutations linked to kindreds with putative hereditary non-polyposis colorectal cancer syndrome
Department of Biosciences, Division of Genetics, University of Helsinki, FIN-00014 Helsinki, Finland
Received January 25, 2002; Accepted March 21, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To date, five mismatch-repair (MMR) genes, MLH1, MSH2, MSH6, MSH3 and PMS2, are known to be involved in human MMR function. Two of those, MLH1 and MSH2, are further the most common susceptibility genes for hereditary non-polyposis colorectal cancer (HNPCC), while MSH3 and PMS2 are seldom (PMS2) or not at all (MSH3 ) reported to be involved in HNPCC. Despite the increasing number of MSH6 germline mutations, their pathogenicity remains questionable, because the mutations are mainly linked to putative HNPCC families lacking the typical clinical and molecular characteristics of the syndrome, such as early age at onset and high microsatellite instability (MSI). High MSI is a consequence of MMR defect, and the pathogenicity of germline mutations in HNPCC is thus linked to malfunction of MMR. To address the question of whether and how MSH6 mutations cause susceptibility to HNPCC, we studied heterodimerization of four MSH6 variants with MSH2, and the functionality of these MutS
complexes in an in vitro MMR assay. All mutations occurred in putative HNPCC patients. Irrespective of the type or the site of the amino acid substitutions, all the variants repaired G
T mismatches to AT as wild-type MSH6 protein. However, the MSH6 protein carrying a mutation in the MSH2/MSH6 interaction region was poorly expressed, suggesting problems in its stability. Our results are clinically relevant, since they demonstrate that under the stable in vitro conditions, when the amounts of the proteins are adequate for repair, the tested MSH6 mutations do not affect repair function. Consequently, while the typical HNPCC syndrome is associated with problems in repair reaction, the pathogenicity of mutations in putative HNPCC families may be linked to other biochemical events. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is associated with malfunction of postreplicative mismatch repair (MMR). While the MMR genes MSH2 and MLH1 account for a majority of HNPCC cases, the number of mutations reported in the MSH6 gene is continually rising (1; http://www.nfdht.nl). Altogether, MSH6 mutations account for 5–10% of kindreds in which MSH2 and MLH1 mutations are excluded (2,3). In contrast to HNPCC families linked to MSH2 or MLH1 mutations, the families associated with MSH6 mutations often display diverse and less typical clinical features. Characteristics of HNPCC, such as early age of onset and high microsatellite instability (MSI) in tumors, are not typical of MSH6 mutation carriers (2–7). Furthermore, they unusually often display carcinomas of the endometrium (2,6–8). The significance of MSH6 in endometrial carcinomas development is emphasized by the observation that lack of MSH6 protein characterizes endometrial but not colon carcinomas in HNPCC (9). Moreover, a majority of epithelial tumors in Msh6-deficient mice have been shown to originate from the uterus (and the skin), and only rarely from the intestine (10).
In a MMR mechanism, the mismatch recognition function is fulfilled by one of the heterodimeric protein complexes, MSH2–MSH6 (MutS) or MSH2–MSH3 (MutSß), dependent on the type of mutation. The MutS complex recognizes base–base mismatches and small insertion–deletion loops (IDL), whereas the MutSß complex recognizes IDLs basically larger than one extrahelical nucleotide (11–14). The MSH6 and MSH3 proteins are shown to be functionally redundant, so that MutS can partially compensate the function of MutSß while MutSß appears to only recognize insertions and deletions (15–17). Because of this redundancy, mutations in MSH6 cause accumulation of base substitutions but less frequently frameshift mutations in microsatellite sequences (15,18). This explains the low MSI in MSH6-deficient tumors. Lack of frameshift mutations, which can easily target repetitive sequences also in tumor suppressor genes and result in their inactivation during tumorigenesis, may further explain the late onset in many MSH6 mutation carriers.
To date, the International Collaborative Group on HNPCC (ICG-HNPCC) has over 30 potentially pathogenic MSH6 mutations in the database (http://www.nfdht.nl). A significant proportion (35%) of them result in a single amino acid substitution, which is difficult to interpret. The pathogenicity of HNPCC mutations is linked to malfunction of MMR. Today, five different genes, MLH1, MSH2, MSH6, MSH3 and PMS2, are definitely known to be involved in human MMR (19). However, the latter two genes are not or are only rarely found to be involved in HNPCC. The late age at onset and the divergent tumor spectrum common in MSH6 kindreds, as well as the low or lack of microsatellite instability frequently observed in the tumors of MSH6 carriers, raises the question of the role of MSH6 germline mutations in HNPCC susceptibility. To address this question, we studied whether the MSH6 proteins carrying selected mutations occurring in putative HNPCC families were deficient in the MMR.
The functionality of the MSH6 variants was analyzed in an in vitro MMR assay. This assay has recently been used to test six MLH1 mutations (20). The results suggested that all the mutations that occurred in typical HNPCC families fulfilling the original Amsterdam criteria for the disease (21) imparted a MMR-deficient phenotype to the altered MLH1 polypeptide, whereas another MLH1 mutation linked to a family with late age at onset was functional in the assay. The four MSH6 missense mutations included in the present study all fulfilled at least two of the following criteria supporting the assumption that the mutations were likely to be pathogenic: the mutation was located in an evolutionarily conserved residue (S144I and G566R), it was not present in healthy control subjects (S144I, G566R, P1087T and P1087R), and MSH2 and MLH1 germline mutations were excluded in the kindred (S144I, G566R, P1087T and P1087R) (2–4,7). However, none of the families in which the four mutations were found fulfilled the Amsterdam (I) criteria (21). The interactions of the four MSH6 variants with normal MSH2 and the functionality of these heterodimers of MSH6 and MSH2 (MutS) will be described.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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None of the studied MSH6 mutations abolishes the MSH2–MSH6 interaction
A combined immunoprecipitation/western blot analysis was used to study the affect of MSH6 mutations on the MSH2–MSH6 interaction. The amount of wild-type MSH2 protein was adjusted to be equal in different extracts, as estimated from denaturing polyacrylamide gel electrophoresis (data not shown) and western blot analysis. As discussed in Materials and Methods, the MSH6 protein is unstable in the absence of its partner, MSH2. By adjusting the MSH2 levels to be equal, we were able to see a decrease in the MSH6 level, which was then not caused by a low level of its partner but rather indicates either a problem in MSH2–MSH6 interaction or an increased instability of the MSH6 variant itself. The purified MutS
complex and the wild-type MutS total extract were used as controls. As shown in Figure 1, the purified MutS, MutS-WT, MutS-S144I, MutS-P1087T and MutS-P1087R all contain similar amounts of MSH2 and MSH6 proteins. However, in the extract MutS-G566R, the amount of MSH6 protein is clearly decreased (Fig. 1, upper panel). There is a reduction even if the recombinant MSH6-G566R baculovirus was used twice as much as normally for transfection and although the amount of MSH2 remains normal. In accordance with this, less MSH6 also co-precipitate less MSH2. However, as is shown in the lower panel of Figure 1, none of the mutations abolished the MSH2–MSH6 interaction, and the other three MutS variants were precipitated in similar amounts as wild-type controls.
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The MSH6 variants are proficient in the in vitro MMR assay
As described above, all the mutant proteins were able to form MSH2–MSH6 heterodimers. In order to find out if the mutations could still be responsible for the MMR defect, we tested the recombinant MutS
variants in the in vitro MMR assay. Since the heteroduplex molecules (GT) are not all repairable, they are always added in excess in the assay, and the repair percentage of the proficient wild-type controls are used as a reference level. As shown in Figure 2, nuclear extract (NE) of MMR-proficient TK6 cells repaired 41% of heteroduplex DNA. NE of the HCT15 (MSH6-/-) cells, which is not able to repair the mismatch, was used as a negative control. The purified MutS and MutS-WT total extract were both able to complement the MSH6-deficient NE of HCT15 cells and repaired approximately 30% of heteroduplex DNA. Interestingly, all the mutant variants were also proficient in this assay. MutS-S144I, MutS-G566R, MutS-P1087T and MutS-P1087R repaired 31%, 30%, 32% and 34%, respectively. The values are an average of two independent experiments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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According to the HNPCC database, the MSH6 gene is the third most common susceptibility gene in the syndrome. To date, MSH6 mutations account for about 10% of all reported mutations, and the number is continuously rising (http://www.nfdht.nl). As distinct from HNPCC families linked to MSH2 or MLH1 mutations, MSH6 mutations are mainly linked to suspected HNPCC families with less typical clinical features (2–7). In particular, the early age of onset and high MSI in tumors, the main hallmarks in HNPCC, are not typical of MSH6 mutation carriers. Instead, they often display later onset, low or lack of MSI, and an atypical tumor spectrum with many endometrial carcinomas (2,4,6). According to a recent report, colorectal carcinomas in MSH6 mutation carriers occur more frequently in the left colon than they do in MLH1 or MSH2 mutation carriers (7).
The first international criteria for the uniform diagnosis of HNPCC were developed in 1991 (21). These criteria, called the Amsterdam (I) criteria, are as follows: (i) histologically verified colorectal cancer in three or more relatives, one of whom is a first-degree relative of the other two; (ii) colorectal cancer involving at least two generations; and (iii) one or more colorectal cancer cases diagnosed before the age of 50 years. However, the phenotypes, the age at onset and the tumor spectrum, vary between the families and even in patients from the same family. Because of that, new less stringent criteria have been developed that accept, besides colorectal carcinomas also extracolonic cancers, notably endometrial carcinomas (modified Amsterdam criteria, Amsterdam criteria II) (22). Sometimes even one colorectal carcinomas patient, when young enough and with a high degree of MSI in tumor tissue, can be an indication of HNPCC (Bethesda guidelines) (23). Since families with MSH6 germline mutations often display atypical hyperplastic lesions and carcinomas of the endometrium, rather than only colorectal carcinomas, they do not usually fulfill the original Amsterdam criteria. Furthermore, the mean age at diagnosis for colorectal carcinomas in MSH6 carriers is reported to be as high as 61 years (3), and for that reason, the families often fail to meet the other criteria too. Nevertheless, the main genetic criterion to assess the involvement of a MSH6 mutation in a hereditary predisposition is its segregation with the cancer phenotype in a family.
The tumors in MSH6 carriers do not usually show instability in dinucleotide repetitive regions typical of tumors in MSH2 and MLH1 mutation carriers. Instead, they more often have unstable mononucleotide repeats (2). The low instability has been explained by the fact that the functions of MSH6 and MSH3 proteins are partially redundant. Studies with human, yeast and murine systems have shown that MutS recognizes both base–base and insertion–deletion mispairs, whereas MutSß mainly recognizes insertion–deletion repair larger than one extrahelical nucleotide (14–17,24). Accordingly, in the human cell in which MSH6 is mutated but MutSß (MSH2–MSH3) is still functional, MSI would be low and limited to mononucleotide tandem repeats. However, a recent report suggests that, irrespective of low MSI typical of tumors in MSH6 mutation carriers, instability of dinucleotide repetitive regions is as frequent as that of mononucleotide repeats (7). In any case, because the target tumor suppressor genes are suspected of being inactivated by frameshift mutations in HNPCC, this would mean that the loss of MSH6 function would not remarkably speed up tumorigenesis, and might explain the late age at onset and possible low penetrance in MSH6 mutation carriers.
The high MSI typical to HNPCC is a consequence of MMR defect in the cell. Consistently, the pathogenicity of HNPCC germline mutations is linked to malfunction of MMR. Given the diverse and sometimes conflicting clinical and molecular data linked to MSH6 mutation carriers and their cancers, we found it important to explore the role of MSH6 in HNPCC predisposition in greater detail. We studied the MSH2–MSH6 heterodimerization of MSH6 variants carrying mutations found from putative HNPCC patients and the functionality of these MutS complexes in the in vitro MMR assay. Using a cell line (HCT15) that has deficient MutS but functional MutSß, and a heteroduplex with a base–base mismatch as a substrate, we could study the functionality of MSH6 variants in MMR. Four potentially pathogenic MSH6 missense mutations were selected from the database, and each of them fulfilled at least two of the following criteria, supporting the assumption that the mutation is pathogenic: the mutated residue is evolutionarily conserved, the mutation is not present in healthy controls and the proband has no MSH2 or MLH1 germline mutations. The MSI status was available for five tumors. One endometrial and one colorectal carcinoma from S144I mutation carriers had the high-MSI phenotypes, whereas the other two tumors had low MSI (4,7). Furthermore, one colorectal carcinoma from the G566R mutation carrier was reported to have high-MSI status (3). However, the immunohistochemistry was not done in that case to confirm that the high-MSI was specifically linked to the inactivation of MSH6 protein and not, for instance, to transcriptional silencing of MLH1, which is frequently associated with somatic MMR defect and high MSI in sporadic colorectal carcinomas. The immunohistochemical analysis of the endometrial carcinoma with the high-MSI and colorectal carcinoma with the low MSI-status did not show loss of MSH6, MSH2 or MLH1 proteins, whereas the high-MSI colon tumor from the S144I mutation carrier was reported to have simultaneous absence of MSH6 and MSH2 stainings, which explains the high MSI in that tumor (7). The reason for high-MSI phenotypes in the other two tumors remains unexplained.
The mutations S144I and G566R were studied in the Saccharomyces cerevisiae-based functional assay and suggested to be loss-of-function and partial loss-of-function mutations, respectively (3). In that assay, the mutations were introduced into the yeast MSH6 gene. The mutant and wild-type plasmids were transformed into the msh3 msh6 double-mutant S. cerevisiae strain, and the rate of reversion of the target frameshift mutation was determined. In the case of the G566R change, the mutant plasmid failed to fully complement the increased rates of accumulation of base substitution and frameshift mutations caused by the MMR defect in the double-mutant strain, while the function of the S144I mutant plasmid was completely lost. In our in vitro MMR assay, which studies the phenotypic consequences of HNPCC mutations in a homologous human MMR system, both variants MutS-S144I and MutS-G566R were, however, as functional as wild-type MutS. The amount of the protein MSH6-G566R, which has the mutation in the MSH2–MSH6 interaction region, was strongly decreased, suggesting either problems in MSH2–MSH6 interaction leading to increased MSH6 degradation, or increased instability in the variant itself. Likewise, in our previous study, all five MLH1 mutations that imparted a MMR-deficient phenotype on the altered MLH1 polypeptide could also be expressed only in amounts significantly lower than the wild-type protein or the MMR-proficient variant (20). However, under stable in vitro circumstances, in which it is possible to produce enough of the MutS-G566R complex for MMR assay, it repaired the mismatch as efficiently as wild-type control. In vivo, the pathogenicity of the G566R mutation may still be linked, not directly to MMR function, but rather to earlier steps, such as protein stability or MSH2–MSH6 interaction.
Codon 1087 is interesting, since it is located in the (C)8 repetitive region in the C-terminal part of the MSH6 polypeptide, which has frequently been used as an MSI marker (2,25). The amino acid is conserved through human, mouse, Escherichia coli and Drosophila melanogaster, but not in S. cerevisiae. Therefore, functional studies of the two mutations P1087T and P1087R are not possible in the yeast assay described above. In our in vitro MMR assay, both of the variants MutS-P1087T and MutS-P1087R were functional as well as the other two tested variants, repairing GT mismatches to AT. Furthermore, according to our co-immunoprecipitation analysis, none of the studied MSH6 mutations abolished the MSH2–MSH6 interaction.
These findings are clinically relevant, since they lead us to suggest that under stable circumstances, as in the in vitro MMR assay, when the amounts of the proteins and MutS heterodimers are adequate for repair, all the four MSH6 mutated polypeptides are as proficient as the wild-type MSH6 protein in MMR function. However, the results do not exclude the possibility that the mutations affect biochemical events preceding the MMR function in vivo – indeed, the discrepancies in results obtained with human in vitro and yeast in vivo systems concerning the mutations S144I and G566R suggests that this is the case. In accordance with the previous study of six MLH1 mutations (20), our results indicate that although the typical HNPCC syndrome is linked with malfunction of MMR, the pathogenicity is a significant proportion of putative HNPCC families combined with less typical clinical features may be mediated through different biochemical events. Further experiments such as analyses of the mutated proteins in selected interactions and studies of the cell lines carrying these mutations will facilitate the definition of functionally important domains of the MMR proteins and the biochemical pathways through which the pathogenic effects of the mutations are mediated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MSH6 mutations and putative HNPCC kindreds
All four MSH6 missense mutations analyzed in the functional assay are included in the public database maintained by ICG-HNPCC (http://www.nfdht.nl). The patients carrying these germline mutations were found not to have germline mutations in MSH2 or MLH1. Furthermore, these mutations did not occur in 185–200 healthy controls (2–4,7). None of the families in which these potentially pathogenic MSH6 mutations were found fulfilled the Amsterdam criteria (Amsterdam criteria I or II) and they were classified as non-HNPCC or suspected HNPCC families (2–4,7,21). The mutations and the phenotypic characteristics of the respective HNPCC families included in the present study are listed in Table 1.
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A missense mutation S144I has been found in four separate families, and has been suggested to be a loss-of-function mutation (3,4,7). Two of the index patients had an endometrial carcinoma (EC) at the age of 45 years and in both families the sister of the index patient had a colorectal cancer (CRC) at the age of 49 years (4,7). In two other families, the index patients had CRC (3,7). One of the ECs was reported to have the low-MSI and another the high-MSI phenotype. In the latter tumor, however, immunohistochemical analysis showed that MSH6 as well as MLH1 and MSH2 stainings were all present, whereas the sister's CRC, which also had high MSI showed absence of both MSH6 and MSH2 stainings (7). Furthermore, one of the index patients with CRC was reported to have low MSI and normal immunohistochemical stainings (7). A mutation G566R was found in siblings who were diagnosed with CRCs at the ages of 62 and 73 years (3). One of the tumors was studied and shown to have the high-MSI phenotype. A proband carrying a mutation P1087T had a CRC at the ages of 57 years and one first-degree relative with a CRC at very late age of onset (90 years) (3). A carrier of the mutation P1087R had a CRC at the age of 37 years. Furthermore, another unspecified member in the family had a CRC at the age of 31 years (2). MSI status was not reported of the tumors linked with the latter two mutations.
Figure 3 shows the locations of the three mutated codons and functional domains in the MSH6 protein. Amino acids S144 and G566 in the N-terminus of the protein are highly conserved through human, mouse and S. cerevisiae (3,4). Moreover, amino acid G566 is located in the MSH2–MSH6 interaction region (26). Amino acid P1087 is conserved through human, mouse, E. coli and Drosophila melanogaster, but not in yeast. P1087 locates in the (C)8 repetitive region in the C-terminal part of the MSH6 polypeptide. This repeat region consisting of eight cytosines has frequently been used as a marker for MSI (2,25).
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Site-directed mutagenesis and production of recombinant baculoviruses
The entire open reading frame of MSH6 was cloned between the BamHI and XhoI sites of the pFastBac1 plasmid (Gibco BRL). Mutations were introduced into MSH6 cDNA using a PCR-based protocol as described previously (20). The primers, fragment sizes, PCR annealing temperatures and cloning sites used in the site-directed mutagenesis are listed in Table 2. The mutated cloned fragments were verified by DNA sequencing (AbiPrism 3100 Genetic Analyzer, Applied Biosystems) (Fig. 4). The recombinant baculovirus constructs were generated from the pFastBac1/MSH6 clones using the Bac-to-Bac system (Gibco BRL) according to the manufacturer's instructions.
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Production of recombinant proteins
The bacmid DNAs containing cDNA inserts encoding wild-type MSH6 (MSH6-WT), mutated MSH6 (MSH6-S144I, MSH6-G566R, MSH6-P1087T and MSH6-P1087R), or wild-type MSH2 (MSH2-WT) were used to transfect Spodoptera frugiperda 9 (Sf9) insect cells (20). For protein extraction, Sf9 cells were co-infected with MSH2 and MSH6 recombinant baculoviruses, since both in vivo studies in mice (10) and in vitro studies in human cells (27,28) have shown that the MSH6 protein (Msh6 in the mouse) is unstable in the absence of its partner MSH2 (Msh2). The total protein extracts including the heterodimeric MutS
were prepared as described previously (20).
Combined MSH2/MSH6 co-immunoprecipitation and western blot analysis
The MSH6 and MSH2 expression levels in the total protein extracts of Sf9 cells were examined by SDS–PAGE. In the co-immunoprecipitation study, 100 µg of each extract was incubated for 16 hours at 4°C rotating with 1 µg of anti-MSH6 monoclonal antibody (Transduction Laboratories, G70220) in a total volume of 1 ml in RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris pH 8.0). After adding 20 µl of protein A/G agarose suspension (Santa Cruz), incubation was continued for a further 3 hours. The immunoprecipitates were recovered by centrifugation (2500 g, 5 minutes), washed three times with 800 µl RIPA buffer and resuspended in 30 µl of 2xSDS sample loading buffer. The immunoprecipitated samples as well as the original protein extracts estimated to contain equal quantities of recombinant wild-type MSH2 protein (MutS-WT extract 3.3 µg, MutS-S144I extract 4.4 µg, MutS-G566R extract 5.0 µg, MutS-P1087T extract 3.0 µg and MutS-P1087R extract 3.7 µg) were loaded on 7.5% SDS–polyacrylamide gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Hybond-P PVDF, Amercham Pharmacia Biotech) for western blot analysis. The membranes were blotted with monoclonal antibodies against MSH6 (Transduction Laboratories, G70220, 0.02 µg/ml) and MSH2 (Calbiochem, MSH2-Ab1, 0.3 µg/ml).
DNA heteroduplex preparation
The circular DNA heteroduplexes containing either an AT or a GT base pair 369 bp downstream from the single-strand nick were prepared as previously described (29–31) using a pGEM phagemid as a template.
In vitro MMR assay
In vitro MMR assay was carried out according to a previous report (20). The repair reaction included 100 ng of heteroduplex DNA and 75 µg of NE of the HCT15 colorectal tumor cell line. This cell line is deficient in repair of base–base mismatches owing to MutS deficiency, since both MSH6 alleles have been inactivated by frameshift mutations (32). The functionality of the mutated MSH6 proteins was studied by complementing HCT15 NE with the total protein extract including overexpressed MutS. Each protein extract was estimated to contain equal quantities of recombinant MSH2 (MutS-WT 16.5 µg, MutS-S144I 22.0 µg, MutS-G566R 25.0 µg, MutS-P1087T 15.0 µg and MutS-P1087R 18.5 µg). MutS purified protein (15.2 µg) and MutS-WT were used as positive controls, and HCT15 NE as a negative control in the assay (MutS purified protein and HCT15 NE were kindly provided by Professor Jiricny). The presence of the GT mismatch makes the pGEM phagemid refractory to cleavage with the restriction endonuclease BglII. The repair reaction converts the GT heteroduplex to the AT homoduplex, so that repair efficiency can be estimated from the amount of phagemid DNA, cleaved by BglII at the conclusion of the repair reaction.
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ACKNOWLEDGEMENTS |
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We thank Professor Josef Jiricny from the University of Zürich for providing us with the TK6 and HCT15 nuclear extracts, original cDNA clones and purified Muts
used in the study. We thank Dr Päivi Peltomäki from the University of Helsinki for a critical reading of the manuscript. This study was supported by grants from the Sigrid Juselius Foundation, the European Commission (QLG1-CT-2000-01230) and the Research Foundation of the University of Helsinki.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Biosciences, Division of Genetics, University of Helsinki, Viikinkaari 5, FIN-00014 Helsinki, Finland. Tel: +358 9 19159073; Fax: +358 9 19159079; Email: minna.nystrom-lahti{at}helsinki.fi
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