Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3.

As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene.

Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells.

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G(17) and A(17)) and a dinucleotide [(CA)(17)] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR(-)) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5' end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.

Detection of common disease-causing mutations in mitochondrial DNA (mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes MTTL1 3243 A>G and myoclonic epilepsy associated with ragged-red fibers MTTK 8344A>G) by real-time polymerase chain reaction.

The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.

Microsatellite instability testing in colorectal carcinoma: choice of markers affects sensitivity of detection of mismatch repair-deficient tumors.

PURPOSE: Microsatellite instability (MSI) is found in 10% to 15% of sporadic colorectal tumors and is usually caused by defects in DNA mismatch repair (MMR). In 1997, a panel of microsatellite markers including mononucleotide and dinucleotide repeats was recommended by a National Cancer Institute workshop on MSI. We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients. EXPERIMENTAL DESIGN: Paraffin sections of normal and tumor tissue from 262 colorectal cancer patients were examined for MSI status by PCR amplification and for MMR protein expression using antibodies against hMLH1, hPMS2, hMSH2, and hMSH6. RESULTS: Twenty-six (10%) of the patients studied had tumors with a high level of MSI (MSI-H). The frequencies of MSI were the same in African-American and Caucasian patients. Each of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats and had loss of MMR protein expression, as did two tumors that had low levels of MSI (MSI-L). These two MSI-L tumors exhibited mutations in mononucleotide repeats only, whereas eight of the other nine MSI-L tumors had mutations in just a single dinucleotide repeat. There was not a statistically significant difference in outcomes between patients whose tumors were MMR-positive or MMR-negative, although there was a slight trend toward improved survival among those with MMR-deficient tumors. CONCLUSIONS: The choice of microsatellite markers is important for MSI testing. Examination of mononucleotide repeats is sufficient for detection of tumors with MMR defects, whereas instability only in dinucleotides is characteristic of MSI-L/MMR-positive tumors.

Induction of a low level of microsatellite instability by overexpression of DNA polymerase Beta.

Microsatellite instability (MSI) is the condition in which high rates of frameshift mutations are observed in short tandem repeat sequences. Mutations in sequences of this type in coding regions of cancer-related genes can contribute to the development of cancer. Although defects in mismatch repair are usually responsible for high levels of MSI, low levels of MSI have been observed in some cancers with no known mismatch repair defects. We have investigated whether overexpression of an error-prone polymerase, polbeta, is sufficient to induce MSI in the presence of mismatch repair. Because overexpression of polbeta has been observed in several types of cancer, we hypothesized that polbeta overexpression might increase genetic instability and, thus, contribute to carcinogenesis. Microsatellite mutation rate analyses were conducted using a drug-resistance reversion assay, where G(17) or A(17) microsatellites were inserted into a plasmid upstream of a neomycin-resistance gene (neo), such that the neo gene was shifted out of frame. When frameshift mutations occur in the microsatellite, the neo gene can be restored, allowing for selection of revertants in G418. Microsatellite-containing plasmids were transfected into telomerase-immortalized normal human fibroblasts (hTERT-1604), where they integrated into the nuclear genome. polbeta-expressing episomal vectors or empty control vectors were then introduced for analysis of the effect of polbeta overexpression on these microsatellites. Mutation rates were determined by fluctuation analysis. Mutation rates in G(17) repeats were elevated for the polbeta transfectants at all levels of overexpression ( approximately 2-fold to >100-fold compared with vector-only controls), with up to a 3-fold increase in mutation rates compared with the vector-only controls in cells with the highest expression. A similar magnitude of elevation in mutation rates was observed for A(17) microsatellites. No difference was observed between vector-only controls and nontransfected cells in either microsatellite sequence. Cells with high polbeta expression showed an approximately 1.5-fold increase in population doubling time and a 2-fold reduction in mitotic index compared with controls. Cells with both modest and high elevations in microsatellite mutation rates had these altered growth properties. These results suggest that polbeta overexpression may affect cell cycle progression and increase genetic instability.

Mutation frequency analysis of mononucleotide and dinucleotide repeats after oxidative stress.

Many tumors exhibit genetic instability at the DNA sequence level in the form of frameshift mutations in simple repeats (microsatellite instability). A high level of microsatellite instability, such as that seen in hereditary nonpolyposis colorectal cancer (HNPCC), arises from defects in the mismatch repair pathway. A low level of microsatellite instability is found in some non-HNPCC-associated cancers, such as those of the breast and lung, and is not attributable to mismatch repair defects. We hypothesized that oxidative DNA damage may be at least partly responsible for the generation of microsatellite mutations in these tumors. We investigated whether oxidative DNA damage can induce microsatellite mutations in mismatch repair-proficient cultured cells. Telomerase-immortalized normal human fibroblasts were stably transfected with a plasmid containing a tk-neo fusion gene, such that the neo coding region was placed out of frame by the presence of an upstream microsatellite sequence. Cells were treated with H(2)O(2) and mutation frequencies were determined for G(17), A(17), and (CA)(17) repeats. Mutation frequencies of mononucleotide repeats in cells with the neo gene in the (+1) reading frame were reduced after treatment. No effect was observed in cells with the mononucleotide repeats in the (-1) reading frame. A small increase in mutation frequency was observed in cells with the (CA)(17) repeat. Our data suggest that diploid human cells may have protective mechanisms that prevent the induction of microsatellite mutations by a short exposure to high levels of oxidative stress.

Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells.

Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.

Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells.

Microsatellites are DNA elements composed of short tandem repeats of 1-5bp. These sequences are particularly prone to frameshift mutation by insertion-deletion loop formation during replication. The mismatch repair system is responsible for correcting these replication errors, and microsatellite mutation rates are significantly elevated in the absence of mismatch repair. We have investigated the effect of varying the number of repeats in a (CA)n microsatellite on mutation rates in cultured mammalian cells proficient or deficient in mismatch repair. We have also compared the relative rates of single-repeat insertions and deletions in these cells. Two plasmid vectors were constructed for each repeat unit number (n=8, 17, and 30), such that the microsatellites, placed upstream of a bacterial neomycin resistance gene (neo), disrupted the reading frame of the gene in the (-1) or (+1) direction. Plasmids were introduced separately into the cells, where they integrated into the cellular genome. Mutation rates were determined by selection of clones with frameshift mutations in the microsatellite that restored the reading frame of the neo gene. We found that mutation rates were significantly higher for (CA)17 and (CA)30 tracts than for (CA)8 tracts in both mismatch repair proficient (mouse) and deficient (human) cells. A mutational bias favoring insertions was generally observed. In both (CA)17 and (CA)30 tracts, single-repeat insertion rates were higher than single-repeat deletion rates with or without mismatch repair; deletions of multiple repeat units (> or =8bp) were observed in these tracts, where as deletions this large were not found in the (CA)8 tract. Single-repeat mutations of both types were made at similar rates in (CA)8 tracts in human mismatch repair deficient (MMR-) cells, but single-repeat insertion rates were higher than single-repeat deletion rates in mouse mismatch repair proficient (MMR+) cells. Results of these direct studies on microsatellite mutations in cultured cells should be useful for refinement of mathematical models for microsatellite evolution.

Mosaicism for an FMR1 gene deletion in a fragile X female.

Most cases of fragile X syndrome result from expansion of CGG repeats in the FMR1 gene; deletions and point mutations of FMR1 are much less common. Mosaicism for an FMR1 full mutation with a deletion or with a normal allele has been reported in fragile X males. Here we report on a fragile X female who is mosaic for an FMR1 full mutation and an intragenic deletion. The patient is a 4-year-old girl with developmental delay, autistic-like behaviors, and significant speech and language abnormalities. Southern blotting demonstrated the presence of a methylated full mutation, a normal allele in methylated and unmethylated forms, and an additional fragment smaller than the normal methylated allele. This result indicates that the patient is mosaic for a full mutation and a deletion, in the presence of a normal allele. By DNA sequence analysis, we mapped the 5' breakpoint 63/65 bp upstream from the CGG repeat region and the 3' breakpoint 86/88 bp downstream of the CGG repeats within the FMR1 gene. The deletion removed 210 bp, including the entire CGG repeat region. The full mutation was inherited from a premutation in the patient's mother. The deletion, which remained methylated at the Eag I and Nru I sites, was probably derived from the full mutation allele. Mosaicism of this type is rare in females with a fragile X mutation but should be kept in mind in the interpretation of Southern blots.

Variation in efficiency of DNA mismatch repair at different sites in the yeast genome.

Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells.

We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.