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PubMed1999

Functional analysis of human MutSalpha and MutSbeta complexes in yeast.

Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA. The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast. Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding. The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.

Alleles↗
PubMed2005

Inverted repeat-stimulated sister-chromatid exchange events are RAD1-independent but reduced in a msh2 mutant.

Inverted repeats (IRs) and trinucleotide repeats (TNRs) that have the potential to form secondary structures in vivo are known to cause genome rearrangements. Expansions of TNRs in humans are associated with several neurological disorders. Both IRs and TNRs stimulate spontaneous unequal sister-chromatid exchange (SCE) in yeast. Secondary structure-associated SCE events occur via double-strand break repair. Here we show that the rate of spontaneous IR-stimulated unequal SCE events in yeast is significantly reduced in strains with mutations in the mismatch repair genes MSH2 or MSH3, but unaffected by a mutation in the nucleotide excision-repair gene RAD1. Non-IR-associated unequal SCE events are increased in both MMR- and rad1-mutant cells; however, SCE events for both IR- and non-IR-containing substrates occur at a higher level in the exo1 background. Our results suggest that spontaneous SCE occurs by a template switching mechanism. Like IRs, TNRs have been shown to generate double-strand breaks (DSBs) in yeast. TNR expansions in mice are MSH2-dependent. Since IR-mediated SCE events are reduced in msh2 cells, we propose that TNR expansion mutations arise when DSBs are repaired using the sister or the homolog as a template.

DNA Damage↗
PubMed2000

Expression of deoxyribonucleic acid repair enzymes during spermatogenesis in mice.

Meiotic recombination during gametogenesis is critical for proper chromosome segregation. However, the participating proteins and mechanics of recombination are not well understood in mammals. DNA repair enzymes play an essential role in both mitosis and meiosis in yeast. The mammalian mismatch repair system consists of homologues of the bacterial MutH, MutL, and MutS proteins. As part of our goal of understanding the function of enzymes that mediate meiotic recombination, we used a reverse transcription-polymerase chain reaction approach to identify germ cell transcripts for the MutL homologue, Pms2, and two members of the MutS family, Msh2 and Msh3. Both the Pms2 and the Msh2 genes were highly expressed in mitotically proliferating spermatogonia, and early in meiotic prophase in the leptotene and zygotene spermatocytes. Thereafter, expression declined in early and mid pachytene spermatocytes, and was negligible in postmeiotic spermatids. In contrast, expression of Msh3 was at its highest level in pachytene spermatocytes. Protein levels were similar to gene expression patterns, and both PMS2 and MSH2 were localized in spermatogonia and spermatocytes. These patterns of expression for genes encoding mismatch repair enzymes are consistent with the proposed roles of the gene products in mismatch repair during both DNA replication and recombination.

Adenosine Triphosphatases↗
PubMed1996

Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.

Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed.

Adenosine Triphosphatases↗
PubMed2000

Arabidopsis MutS homologs-AtMSH2, AtMSH3, AtMSH6, and a novel AtMSH7-form three distinct protein heterodimers with different specificities for mismatched DNA.

Arabidopsis mismatch repair genes predict MutS-like proteins remarkably similar to eukaryotic MutS homologs-MSH2, MSH3, and MSH6. A novel feature in Arabidopsis is the presence of two MSH6-like proteins, designated AtMSH6 and AtMSH7. Combinations of Arabidopsis AtMSH2 with AtMSH3, AtMSH6, or AtMSH7 proteins-products of in vitro transcription and translation-were analyzed for interactions by analytical gel filtration chromatography. The AtMSH2 protein formed heterodimers with AtMSH3, AtMSH6, and AtMSH7, but no single proteins formed homodimers. The abilities of the various heterodimers to bind to mismatched 51-mer duplexes were measured by electrophoretic mobility-shift assays. Similar to the behavior of the corresponding human proteins, AtMSH2*AtMSH3 heterodimers bound "insertion-deletion" DNA with three nucleotides (+AAG) or one nucleotide (+T) looped out much better than they bound DNA with a base/base mispair (T/G), whereas AtMSH2*AtMSH6 bound the (+T) substrate strongly, (T/G) well, and (+AAG) no better than it did a (T/A) homoduplex. However, AtMSH2*AtMSH7 showed a different specificity: moderate affinity for a (T/G) substrate and weak binding of (+T). Thus, AtMSH2*AtMSH7 may be specialized for lesions/base mispairs not tested or for (T/G) mispairs in special contexts.

Adenosine Triphosphatases↗
PubMed1997

Altered replication and inverted repeats induce mismatch repair-independent recombination between highly diverged DNAs in yeast.

Replication, DNA organization, and mismatch repair (MMR) can influence recombination. We examined the effects of altered replication due to a mutation in the polymerase delta gene, long inverted repeats (LIRs) in motifs similar to those in higher eukaryotes, and MMR on intrachromosomal recombination between highly diverged (28%) truncated genes in Saccharomyces cerevisiae. A combination of altered replication and an LIR increased recombination up to 700-fold, while each alone led to a 3- to 20-fold increase. Homeologous recombination was not altered by pms1, msh2, and msh3 mismatch repair mutations. Similar to our previous observations for replication slippage-mediated deletions, there were > or = 5-bp identical runs at the recombination breakpoints. We propose that the dramatic increase in recombination results from enhancement of the effects of altered replication by the LIR, leading to recombinationally active initiating structures. Such interactions predict replication-related, MMR-independent genome changes.

Base Sequence↗
PubMed1997

Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins.

A change in the number of base pairs within a coding sequence can result in a frameshift mutation, which almost invariably eliminates the function of the encoded protein. A frameshift reversion assay with Saccharomyces cerevisiae that can be used to examine the types of insertions and deletions that are generated during DNA replication, as well as the editing functions that remove such replication errors, has been developed. Reversion spectra have been obtained in a wild-type strain and in strains defective for defined components of the postreplicative mismatch repair system (msh2, msh3, msh6, msh3 msh6, pms1, and mih1 mutants). Comparison of the spectra reveals that yeast mismatch repair proteins preferentially remove frameshift intermediates that arise in homopolymer tracts and indicates that some of the proteins have distinct substrate or context specificities.

Adenosine Triphosphatases↗
PubMed1998

Interactions of human hMSH2 with hMSH3 and hMSH2 with hMSH6: examination of mutations found in hereditary nonpolyposis colorectal cancer.

Mutations in the human mismatch repair protein hMSH2 have been found to cosegregate with hereditary nonpolyposis colorectal cancer (HNPCC). Previous biochemical and physical studies have shown that hMSH2 forms specific mispair binding complexes with hMSH3 and hMSH6. We have further characterized these protein interactions by mapping the contact regions within the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers. We demonstrate that there are at least two distinct interaction regions of hMSH2 with hMSH3 and hMSH2 with hMSH6. Interestingly, the interaction regions of hMSH2 with either hMSH3 or hMSH6 are identical and there is a coordinated linear orientation of these regions. We examined several missense alterations of hMSH2 found in HNPCC kindreds that are contained within the consensus interaction regions. None of these missense mutations displayed a defect in protein-protein interaction. These data support the notion that these HNPCC-associated mutations may affect some other function of the heterodimeric complexes than simply the static interaction of hMSH2 with hMSH3 or hMSH2 with hMSH6.

Binding Sites↗
PubMed1999

Mismatch repair processing of carcinogen-DNA adducts triggers apoptosis.

The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSalpha and hMutSbeta efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1999

Removal of frameshift intermediates by mismatch repair proteins in Saccharomyces cerevisiae.

Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Isolation and characterization of point mutations in mismatch repair genes that destabilize microsatellites in yeast.

The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2 mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis.

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

Adenosine Triphosphatases↗
PubMed1998

Mismatch repair genes and mononucleotide tracts as mutation targets in colorectal tumors with different degrees of microsatellite instability.

Microsatellite instability occurs in 15% of colorectal carcinomas and may be due to replication errors (RER). The pattern of instability--'severe' vs 'mild'--and the tumorigenic pathway, as reflected by the involvement of functionally important genes, may vary according to the underlying gene(s). We defined 'mild' RER as mono- or tetranucleotide repeat instability in the absence of widespread instability at dinucleotide repeats and studied 15 colorectal tumors with this phenotype for mutations in the DNA mismatch repair genes MSH2, MLH1, MSH3, and MSH6. No mutations were found, suggesting that these genes were not implicated. We then compared colorectal cancers with 'mild' RER (n = 15), and those with 'severe' RER without (n = 11) or with (n = 22) detectable mutations in MSH2 or MLH1 to assess the involvement of mononucleotide repeats contained in the coding regions of MSH3, MSH6, BAX, and TGFbeta RII. The combined mutation rates of the above mentioned loci varied significantly between the three groups of tumors, being 0%, 25% and 52%, respectively. Furthermore, the individual genes showed specific patterns of involvement; for example, among tumors with 'severe' RER, TGFbeta RII displayed uniformly high mutation rates while MSH3, MSH6, and BAX were more frequently altered in tumors that also showed MSH2 or MLH1 mutations. Our findings suggest that different subcategories exist among unstable tumors, defined by the RER pattern on the one hand and tumorigenic pathway on the other, and structural changes of MSH2 and MLH1 are likely to explain only a proportion of these cases.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Distinct clinical features and outcomes of gastric cancers with microsatellite instability.

Microsatellite instability (MSI) is a hallmark of the DNA mismatch repair deficiency that is one of the pathways of gastric carcinogenesis. Clinicopathologic characteristics of MSI+ gastric cancers remain unclear. To determine the correlation between MSI status and clinical features, we analyzed 327 consecutive gastric cancers for the occurrence of MSI in the BAT-26 marker. Because it has been proven that MSI at BAT-26 reflects the MSI+ phenotype, cancers with alteration at BAT-26 were categorized as having the MSI+ phenotype. The expressions of hMLH1, hMSH2, p53, MUC1, MUC2, and CEA were evaluated immunohistochemically using the tissue array method. The MSI+ phenotype was found in 9.5% (31/327) of gastric cancers examined. MSI+ gastric cancers were significantly associated with older age, antral location, Borrmann's gross Type II, intestinal subtype, lower prevalence of lymph node metastasis, and lower pTNM stage (P <.05). By multivariate logistic regression, MSI+ gastric cancers had a lower prevalence of lymph node metastasis independent of tumor invasion (P <.001). MSI+ gastric cancers displayed frequent frameshift mutations of transforming growth factor-beta type II receptor (90.3%), BAX (61.3%), hMSH3 (38.7%), and E2F4 (61.3%) genes and diminished hMLH1 (24/31) or hMSH2 (4/31) expressions. The MSI+ phenotype correlated with patient survival in advanced gastric carcinoma (P =.046). In conclusion, MSI+ phenotype in gastric cancers was found to have distinct clinicopathologic characteristics and to be predictive of a favorable outcome in advanced carcinoma.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Microsatellite instability in synchronous gastric carcinomas.

Synchronous gastric carcinomas are found in 4% to 10% of all gastric carcinomas, and the tumor multiplicity is believed to be related to genetic predisposition. To investigate the role of mismatch repair error in synchronous gastric carcinomas, we analyzed the microsatellite instability (MSI) status of 101 cancers from 48 gastrectomy specimens and compared them with 149 solitary gastric carcinomas. Multiple synchronous gastric carcinomas are characterized by slightly older age, predominance in males, early stage and lower lymph node metastasis. Among the 48 cases, 8 (18 lesions) were associated with a gastric adenoma (type I) and 40 (83 lesions) were not associated with a gastric adenoma (type II). The MSI+ rate was 50% in the type I and 8.4% in the type II synchronous gastric carcinomas (p < 0.001), while that of solitary gastric carcinomas was 9.4%. In addition, the frameshift mutation rates of the TGF-betaRII, BAX and hMSH3 genes in the type I synchronous carcinomas were higher than those in the type II synchronous carcinomas. These findings indicate that a defect in the mismatch repair system might play a role in the carcinogenesis of a minor subset of multiple gastric carcinomas associated with adenomas.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Screening for defective DNA mismatch repair in stage II and III colorectal cancer patients.

BACKGROUND & AIMS: Colorectal cancers associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome usually present in younger patients, show loss of mismatch repair (MMR) gene expression, and exhibit microsatellite instability (MSI). About 12% of sporadic colorectal cancers also show MMR loss and MSI. The aims of this study were to evaluate MMR loss and MSI in relation to patient age, sex, tumor stage, and site in the large bowel. METHODS: Tissue microarrays were created from 1020 stage II and III colorectal cancer cases and immunohistochemical staining performed to detect expression of the 2 major MMR proteins, hMLH1 and hMSH2. MSI was determined using the BAT-26 mononucleotide repeat. RESULTS: Ten percent of tumors showed loss of hMLH1 expression and 1.2% showed loss of hMSH2 expression. hMLH1 loss was more frequent in women (P < .001), older patients (P = .004), earlier stage tumors (P = .0001), and proximal colon tumors ( P < .0001). In contrast, tumors showing hMSH2 loss were more frequent in younger (P < .001), male (P = .05) patients and were distributed evenly between the proximal colon and distal colon/rectum. Eleven percent of tumors were MSI+ and these showed similar age, sex, stage, and site characteristics as tumors with hMLH1 loss. Discordance between MMR loss and MSI+ was found in 24 of 983 (2.4%) tumors. Of the 231 patients aged <60 years at diagnosis, 12 (5.2%) showed loss of hMLH1 and 8 (3.5%) showed loss of hMSH2. CONCLUSIONS: Routine immunohistochemical screening for MMR loss in younger colorectal cancer patients may provide a useful, first-step screening tool for the population-based detection of HNPCC.

Adaptor Proteins, Signal Transducing↗