Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

Mutation of a mutL homolog in hereditary colon cancer

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.     

Molecular mechanisms of carcinogenesis: the role of systems of DNA repair

The initiation step of the carcinogenic process consists in an alteration of genes playing a central role in the cellular life. The next steps of promotion and progression result from anomalies in the response to growth factors, to hormones and/or from the action of tumor promotors leading to cellular hyperplasia. This process generally leads to genetic instability of the initiated cell which in turn allows selection of malignant and invasive clones. The production of DNA damage by physical or chemical agents is dose-dependent. The error-free enzymatic repair processes including excision resynthesis of base damage or of altered nucleotides allow the restitution of intact DNA. The error-prone repair systems permit survival in association with transmissible alterations (genes and chromosomal mutations). Absence of repair leads to cytotoxicity, programmed cell death or disruption of cell cycle control leading to a pretumoral state. The major role played by mutations in the initiation of carcinogenesis is evidenced by the existence of genetic syndromes associated to hypersensitivity to genotoxic agents, defects in DNA repair capacity, anomalies in the expression of certain genes (including the tumor suppressor p53 gene, etc.) and an elevated predisposition to cancer. Xeroderma pigmentosum which is defective in excision-repair, ataxia telangiectasia and Fanconi anemia which are associated to anomalies in DNA recombination and the familial type of colon cancer HPNCP due to inefficient mismatch repair constitute paradigm for this fundamental notion. Alterations in the capacity to rejoin radiation induced DNA strand breaks appears to be associated to over-reactions to radiotherapy of cancer patients. Also the predisposition to develop secondary thyroid tumors following treatment of a primary cancer in childhood seems to involve the same defect. The existence in the general population of heterozygotes for such DNA repair genes should be taken into account for risk evaluation to therapeutic and environmental exposures.     

Proteins that participate in nucleotide excision repair of DNA in mammalian cells

The most versatile strategy for repair of damage to DNA, and the main process for repair of UV-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease.     

Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.     

Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin

The c-Abl nonreceptor tyrosine kinase and the c-Jun NH2-terminal kinase (JNK/stress-activated protein kinase) are activated during the injury response to the DNA-damaging agent cisplatin. Loss of DNA mismatch repair activity results in resistance to cisplatin in human cancer cells, suggesting that the mismatch repair proteins function as a detector for cisplatin DNA adducts. To identify signaling pathways activated by this detector, we investigated the effect of the loss of DNA mismatch repair function on the ability of cisplatin to activate the JNK and c-Abl kinases. The results demonstrate that cisplatin activates JNK kinase 3.8 +/- 0.2-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that activation of c-Abl is completely absent in the DNA mismatch repair-deficient cells. Furthermore, the results show that cisplatin-induced activation of JNK occurs through a stress-activated protein kinase/extracellular signal-regulated kinase kinase 1-independent mechanism. We conclude that activation of JNK and c-Abl by cisplatin is in part dependent upon the integrity of DNA mismatch repair function, suggesting that these kinases are part of the signal transduction pathway activated when mismatch repair proteins recognize cisplatin adducts in DNA.     

Genetic polymorphisms in carcinogen metabolism and their association to hereditary nonpolyposis colon cancer

BACKGROUND & AIMS: The phenotype of hereditary nonpolyposis colorectal cancer shows interfamilial and intrafamilial variation even in the presence of identical predisposing mutations, suggesting the existence of additional phenotype determinants. The modifying role of genetic polymorphisms in loci involved in carcinogen metabolism was studied. METHODS: We focused on colon cancers from kindreds sharing one of two predisposing mutations (mutation 1 or 2) in the mismatch repair gene MLH1 (78 and 14 tumors, respectively). Polymorphisms in N-acetyltransferase 1 (NAT1) and glutathione S-transferase (GST) M1 and GSTT1 were investigated. RESULTS: The NAT1 allele 10 was associated with lower median age at diagnosis in both groups. In mutation 1 group, the NAT1 allele 10 was a risk factor for distal tumor location, both alone (P = 0.028) and combined with the GSTT1-positive genotype (P = 0.008). On the other hand, the combined null genotype of GSTM1 and GSTT1 was associated with proximal tumors. Associations with tumor location were not observed in patients with mutation 2, probably reflecting a small sample size. CONCLUSIONS: The results suggest that genetic polymorphisms in carcinogen metabolism modify the age of onset and tumor location in individuals with inherited deficiency of DNA mismatch repair.     

Androgen receptor and mating-induced fos immunoreactivity are co-localized in limbic and midbrain neurons that project to the male rat medial preoptic area

The repair of DNA damage protects the genome of the cell from the insults of cancer causing agents. This was originally demonstrated in individuals with the rare genetic disease, xeroderma pigmentosum, the prototype of cancer genes, and subsequently in the relationship of mismatch repair to colon cancer. Recent studies suggest that individuals with less dramatic reductions in the capacity to repair DNA damage are observed at polymorphic frequency and these individuals have an increased susceptibility to several types of cancer. Screening of individuals for DNA sequence variation in the exons of 9 DNA repair genes has resulted in identification of 15 different polymorphic amino acid substitution variants. Although the studies to relate these variants to reduced DNA repair capacity and cancer status have not been completed, the available information is sufficient to suggest that DNA repair genes should be incorporated into molecular epidemiology and cancer susceptibility studies. The availability of molecular epidemiology data presents exciting opportunities for refinement of risk estimation models and identification of individuals at increased risk of disease, with resultant opportunities for effective surveillance and early intervention and treatment. The opportunities to acquire susceptibility data are associated with possible perils for establishment of regulations for permissible exposures to carcinogenic agents and also stigmatization of 'at risk' individuals that may result in decreased access to employment opportunities and health care.     

Molecular biology of colorectal cancer

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. This cancer develops as a result of the pathologic transformation of normal colonic epithelium to an adenomatous polyp and ultimately an invasive cancer. The multistep progression requires years and possibly decades and is accompanied by a number of recently characterized genetic alterations. Mutations in two classes of genes, tumor-suppressor genes and proto-oncogenes, are thought to impart a proliferative advantage to cells and contribute to development of the malignant phenotype. Inactivating mutations of both copies (alleles) of the adenomatous polyposis coli (APC) gene--a tumor-suppressor gene on chromosome 5q--mark one of the earliest events in colorectal carcinogenesis. Germline mutation of the APC gene and subsequent somatic mutation of the second APC allele cause the inherited familial adenomatous polyposis syndrome. This syndrome is characterized by the presence of hundreds to thousands of colonic adenomatous polyps. If these polyps are left untreated, colorectal cancer develops. Mutation leading to dysregulation of the K-ras protooncogene is also thought to be an early event in colon cancer formation. Conversely, loss of heterozygosity on the long arm of chromosome 18 (18q) occurs later in the sequence of development from adenoma to carcinoma, and this mutation may predict poor prognosis. Loss of the 18q region is thought to contribute to inactivation of the DCC tumor-suppressor gene. More recent evidence suggests that other tumor-suppressor genes--DPC4 and MADR2 of the transforming growth factor beta (TGF-beta) pathway--also may be inactivated by allelic loss on chromosome 18q. In addition, mutation of the tumor-suppressor gene p53 on chromosome 17p appears to be a late phenomenon in colorectal carcinogenesis. This mutation may allow the growing tumor with multiple genetic alterations to evade cell cycle arrest and apoptosis. Neoplastic progression is probably accompanied by additional, undiscovered genetic events, which are indicated by allelic loss on chromosomes 1q, 4p, 6p, 8p, 9q, and 22q in 25% to 50% of colorectal cancers. Recently, a third class of genes, DNA repair genes, has been implicated in tumorigenesis of colorectal cancer. Study findings suggest that DNA mismatch repair deficiency, due to germline mutation of the hMSH2, hMLH1, hPMS1, or hPMS2 genes, contributes to development of hereditary nonpolyposis colorectal cancer. The majority of tumors in patients with this disease and 10% to 15% of sporadic colon cancers display microsatellite instability, also know as the replication error positive (RER+) phenotype. This molecular marker of DNA mismatch repair deficiency may predict improved patient survival. Mismatch repair deficiency is thought to lead to mutation and inactivation of the genes for type II TGF-beta receptor and insulin-like growth-factor II receptor. Individuals from families at high risk for colorectal cancer (hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis) should be offered genetic counseling, predictive molecular testing, and when indicated, endoscopic surveillance at appropriate intervals. Recent studies have examined colorectal carcinogenesis in the light of other genetic processes. Telomerase activity is present in almost all cancers, including colorectal cancer, but rarely in benign lesions such as adenomatous polyps or normal tissues. Furthermore, genetic alterations that allow transformed colorectal epithelial cells to escape cell cycle arrest or apoptosis also have been recognized. In addition, hypomethylation or hypermethylation of DNA sequences may alter gene expression without nucleic acid mutation.     

DNA repair: knockouts still mutating after first round

Recent studies have investigated whether particular DNA repair pathways are involved in the somatic hypermutation mechanism that increases antibody diversity. The primary mutation mechanism still functions in mice carrying knockouts of all repair genes examined, but mismatch repair defects affect the final outcome.     

Enhanced intestinal adenomatous polyp formation in Pms2-/-;Min mice

Analysis of two human familial cancer syndromes, hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis, indicates that mutations in either one of four DNA mismatch repair gene homologues or the adenomatous polyposis coli (APC) gene, respectively, are important for the development of colorectal cancer. To further investigate the role of DNA mismatch repair in intestinal tumorigenesis, we generated mice with mutations in both Apc and the DNA mismatch repair gene, Pms2. Whereas Pms2-deficient mice do not develop intestinal tumors, mice deficient in Pms2 and heterozygous for Min, an allele of Apc, develop approximately three times the number of small intestinal adenomas and four times the number of colon adenomas relative to Min and Pms2+/-;Min mice. Although Pms2 deficiency clearly increases adenoma formation in the Min background, histological analysis indicated no clear evidence for progression to carcinoma.     

Correction of hypermutability, N-methyl-N'-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6

The human DNA mismatch repair genes hMSH2 and hMSH6 encode the proteins that, together, bind to mismatches to initiate repair of replication errors. Human tumor cells containing mutations in these genes have strongly elevated mutation rates in selectable genes and at microsatellite loci, although mutations in these genes cause somewhat different mutator phenotypes. These cells are also resistant to killing by certain drugs and are defective in mismatch repair. Because the elevated mutation rates in these cells may lead to mutations in additional genes that are causally related to the other defects, here we attempt to establish a cause-effect relationship between the hMSH2 and hMSH6 gene mutations and the observed phenotypes. The endometrial tumor cell line HEC59 contains mutations in both alleles of hMSH2. The colon tumor cell line HCT15 contains mutations in hMSH6 and also has a sequence change in a conserved region of the coding sequence for DNA polymerase delta, a replicative DNA polymerase. We introduced human chromosome 2 containing the wild-type hMSH2 and hMSH6 genes into HEC59 and HCT15 cells. Introduction of chromosome 2 to HEC59 cells restored microsatellite stability, sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine treatment, and mismatch repair activity. Transfer of chromosome 2 to HCT15 cells also reduced the mutation rate at the HPRT locus and restored sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine treatment and mismatch repair activity. The results demonstrate that the observed defects are causally related to mutations in genes on chromosome 2, probably hMSH2 or hMSH6, but are not related to sequence changes in other genes, including the gene encoding DNA polymerase delta.     

Drug-induced sleep disorders]

Mismatch repair genes are involved in increasing the fidelity of replication by specific repair of DNA polymerase incorporation errors. In Escherichia coli, the best studied mismatch repair (MMR) pathway is the methyl-directed long patch repair system which is mediated by three gene products; MutS, MutL and MutH. These are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding regions of MMR genes result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair.     

Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Fluconazole susceptibility of Candida isolates from oropharyngeal candidosis

New technologies in molecular biology will allow the improvement of screening, diagnosis and prognosis of colorectal cancer patients. For example the determination of germline mutation in APC or in mismatch repair genes in patient with familial adenomatous polyposis or with HNPCC is now possible. The clinical surveillance can be restricted to the patients with these germline defects. More over the knowledge of somatic genetic alterations in colorectal cancer cells seems to be useful in the determination of prognosis of these patients or in order to predict the chemotherapy response.     

Spontaneous intestinal carcinomas and skin neoplasms in Msh2-deficient mice

Hereditary nonpolyposis colorectal cancer is associated with defects in DNA mismatch repair. Here, we characterize tumor susceptibility of the recently described Msh2-deficient mouse model. Within the first year of observation, all homozygous mice succumbed to disease, with lymphomas observed in at least 80% of the cases. The majority (70%) of animals 6 months or older developed intestinal neoplasms associated with APC inactivation. Microsatellite instability was more common in carcinomas than in adenomas, but uncommon in normal tissues. Some animals (7%) developed a variety of skin neoplasms analogous to the Muir-Torre syndrome. Msh2-/- mice implicate a direct role for mismatch repair in several neoplasms with striking phenotypic similarities to humans.     

Reduced COX-2 protein in colorectal cancer with defective mismatch repair

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.