Mismatch Repair Defective Cells Research Articles

Calorie restriction promotes genome stability in mismatch repair defected cells during aging (LB118)

Mismatch repair is a DNA repair system which is critical for the maintenance of genome stability. Defects in mismatch repair have been linked to colorectal and sporadic cancers. Calorie restriction (CR) has been shown to extend life span in various organisms and reduce tumor incidence in mammals. By using Saccharomyces cerevisiae model system for CR and aging studies, mismatch repair defective cells (msh2Δ, msh3Δ, msh6Δ, msh2Δmsh3Δ, msh2Δmsh6Δ and msh3Δmsh6Δ) were cultured with normal (2% glucose) and CR (0.5% glucose) medium. Here we demonstrate that CR containing 0.5% glucose compared to normal treatment (2.0% glucose) can extend life span and promote HOM3 gene stability in all MMR‐defected cells during aging process in yeast. Although the levels of reactive oxygen species (ROS) were significantly increased during aging and CR dramatically reduce the levels of ROS, the levels of ROS had no positive correlation with HOM3 gene mutation rates by comparing those in wild type cells and MMR‐defected cells. The underlying mechanisms need to be further investigated.

Mutation Rates, Spectra, and Genome-Wide Distribution of Spontaneous Mutations in Mismatch Repair Deficient Yeast

DNA mismatch repair is a highly conserved DNA repair pathway. In humans, germline mutations in hMSH2 or hMLH1, key components of mismatch repair, have been associated with Lynch syndrome, a leading cause of inherited cancer mortality. Current estimates of the mutation rate and the mutational spectra in mismatch repair defective cells are primarily limited to a small number of individual reporter loci. Here we use the yeast Saccharomyces cerevisiae to generate a genome-wide view of the rates, spectra, and distribution of mutation in the absence of mismatch repair. We performed mutation accumulation assays and next generation sequencing on 19 strains, including 16 msh2 missense variants implicated in Lynch cancer syndrome. The mutation rate for DNA mismatch repair null strains was approximately 1 mutation per genome per generation, 225-fold greater than the wild-type rate. The mutations were distributed randomly throughout the genome, independent of replication timing. The mutation spectra included insertions/deletions at homopolymeric runs (87.7%) and at larger microsatellites (5.9%), as well as transitions (4.5%) and transversions (1.9%). Additionally, repeat regions with proximal repeats are more likely to be mutated. A bias toward deletions at homopolymers and insertions at (AT)n microsatellites suggests a different mechanism for mismatch generation at these sites. Interestingly, 5% of the single base pair substitutions might represent double-slippage events that occurred at the junction of immediately adjacent repeats, resulting in a shift in the repeat boundary. These data suggest a closer scrutiny of tumor suppressors with homopolymeric runs with proximal repeats as the potential drivers of oncogenesis in mismatch repair defective cells.

Deficiency in DNA mismatch repair increases the rate of telomere shortening in normal human cells

DNA mismatch repair (MMR) is essential for genome stability and inheritance of a mutated MMR gene, most frequently MSH2 or MLH1, results in cancer predisposition known as Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC). Tumors that arise through MMR deficiency show instability at simple tandem repeat loci (STRs) throughout the genome, known as microsatellite instability (MSI). The STR instability is dominated by errors that accumulate during replication in the absence of effective MMR. In this study we show that there is a high level of instability within telomeric DNA with a tendency toward deletions in tumor-derived MMR defective cell lines. We downregulated MSH2 expression in a normal fibroblast cell line and isolated four clones, with differing levels of MSH2 depletion. The telomere-shortening rate was measured at the Xp/Yp, 12q, and 17p telomeres in the MSH2 depleted and three control clones. Interestingly the mean telomere-shortening rate in the clones with MSH2 depletion was significantly greater than in the control clones. This is the first demonstration that MSH2 deficiency alone can lead to accelerated telomere shortening in normal human cells.

Telomere instability detected in sporadic colon cancers, some showing mutations in a mismatch repair gene.

Human telomeres are essential for genome stability and are composed of long simple tandem repeat arrays (STRs), comprising the consensus TTAGGG repeat interspersed, at the proximal end, with sequence-variant repeats. While the dynamics of telomere attrition through incomplete replication has been studied extensively, the effects on telomeres of error-prone DNA repair processes, known to affect other STRs, are poorly understood. We have compared the TTAGGG and sequence-variant interspersion patterns in the proximal 720 bp of telomeres in colon cancer and normal DNA samples. The frequency of telomere mutations was 5.8% per allele in a randomly collected panel of sporadic colon cancers, showing that telomere mutations occur in vivo. The mutation frequency rose to 18.6% per allele in sporadic tumours that exhibit instability at the polyA tract in the TGFbetaRII gene and to 35% per allele in tumours with somatic mutations in the hMSH2 gene. The majority of the characterized mutations resulted in the loss of one or a few repeats. If the mutation spectrum and frequency described here is reiterated in the rest of the array, there is the potential for extensive telomere destabilization especially in mismatch repair-defective cells. This may in turn lead to a greater requirement for telomere length maintenance earlier in tumourigenesis.

Human MRE11 is inactivated in mismatch repair-deficient cancers.

Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia-Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia-Telangiectasia-like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild-type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair-deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11--NBS1--RAD50 complex, an impaired S-phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation-induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair-defective cells and suggest its impairment may contribute to the development of colorectal cancer.

Human MutY Homolog, a DNA Glycosylase Involved in Base Excision Repair, Physically and Functionally Interacts with Mismatch Repair Proteins Human MutS Homolog 2/Human MutS Homolog 6

Adenines mismatched with guanines or 7,8-dihydro-8-oxo-deoxyguanines that arise through DNA replication errors can be repaired by either base excision repair or mismatch repair. The human MutY homolog (hMYH), a DNA glycosylase, removes adenines from these mismatches. Human MutS homologs, hMSH2/hMSH6 (hMutSalpha), bind to the mismatches and initiate the repair on the daughter DNA strands. Human MYH is physically associated with hMSH2/hMSH6 via the hMSH6 subunit. The interaction of hMutSalpha and hMYH is not observed in several mismatch repair-defective cell lines. The hMutSalpha binding site is mapped to amino acid residues 232-254 of hMYH, a region conserved in the MutY family. Moreover, the binding and glycosylase activities of hMYH with an A/7,8-dihydro-8-oxo-deoxyguanine mismatch are enhanced by hMutSalpha. These results suggest that protein-protein interactions may be a means by which hMYH repair and mismatch repair cooperate in reducing replicative errors caused by oxidized bases.

Comparison of spontaneous and adaptive mutation spectra in yeast

Adaptive mutations occur in nongrowing populations of cells to overcome strong, nonlethal selection conditions. Several models have been proposed for the molecular mechanism(s) for this phenomenon inEscherichia coli, but the mechanisms involved in adaptive mutagenesis in the yeastSaccharomyces cerevisiae are largely unknown. We present here a comparison of the mutational spectra of spontaneous and adaptive frameshift reversion events in yeast. In contrast to results fromE. coli, we find that the mutational spectrum of adaptive mutations inS. cerevisiae is not similar to that seen in mismatch repair defective cells, but rather resembles the spontaneous mutational events that occur during normal growth.

Cellular resistance and hypermutability in mismatch repair-deficient human cancer cell lines following treatment with methyl methanesulfonate

Resistance to the cytotoxic effects of S N1 alkylating agents such as N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and N-methyl- N-nitrosourea (MNU) is well established in mismatch repair-defective cells, however, little is known about the cellular response to S N2 alkylating agents in these cells. Here we describe the cytotoxic response and the mutagenic response at the hypoxanthine-guanine phosphoribosyl transferase ( HPRT) locus to the S N2 alkylating agent methyl methanesulfonate (MMS) in human cancer cell lines defective in mismatch repair (MMR). Our findings suggest that cytotoxicity to MMS is mediated through MMR, as indicated by an increased resistance to MMS in MMR-deficient cells. Cells in which specific MMR-gene defects were complemented by chromosome transfer were generally more sensitive to the cytotoxic effects of MMS. Additionally, the induced mutant frequency at HPRT following exposure to MMS is significantly increased in MMR-deficient lines. These findings suggest that resistance to S N2 alkylation damage is mediated by MMR genes, and that resistance to such damage in MMR-defective cells correlates with an increase in genomic mutations. The results are consistent with the hypothesis that abasic sites may be substrates for repair involving MMR-gene products in human cells.

A direct role for DNA polymerase III in adaptive reversion of a frameshift mutation in Escherichia coli

The sequences of adaptive reversions of a lac frameshift mutation in Escherichia coli resemble DNA polymerase errors, and the adaptive reversions decrease in strains with an antimutator DNA polymerase III (PolIII) allele. The latter finding could imply that DNA PolIII itself makes adaptive mutations. Alternatively, normal DNA PolIII errors could saturate post-synthesis mismatch repair during adaptive mutation. If so, the antimutator strain would produce fewer adaptive mutations because it possesses greater capacity for mismatch repair which could correct errors made by a polymerase other than DNA PolIII. Mismatch repair capacity is limited specifically during adaptive mutation, necessitating a test of this indirect model. This indirect model is ruled out here by the observation that the antimutator PolIII allele decreases adaptive mutation even in mismatch repair-defective cells. This supports a direct role for DNA PolIII in recombination-dependent adaptive mutation.

Processing of O6-methylguanine by mismatch correction in human cell extracts

Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutSα mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase δ and/or ϵ. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.

Mutations produced by DNA polymerase III holoenzyme of Escherichia coli after in vitro synthesis in the absence of single‐strand binding protein

Single-stranded plasmid DNA, containing the mnt gene, was replicated in vitro with DNA polymerase III holoenzyme. Escherichia coli mutH bacteria, defective in mismatch repair, were transformed with the products of in vitro synthesis. Mutations in mnt were readily identified and 33 out of 65 isolates were single base changes including transition, transversion and frameshift mutations. The remaining 32 isolates were deletions of apparently random length and substitutions (deletion/insertions). The intergenic deletions as well as the transition and frameshift mutations were identical to those previously isolated from mismatch repair-defective cells in vivo.