The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.     

Clinical and Molecular Characterization of Brazilian Patients Suspected to Have Lynch Syndrome

Lynch syndrome (LS) accounts for 3–5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.     

In vitro studies of DNA mismatch repair proteins

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O⁶meG:T mismatches, the purification of recombinant native human MutSα (MSH2–MSH6) and MutLα (MLH1–PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.     

The Mutational Spectrum of Lynch Syndrome in Cyprus

Lynch syndrome is the most common form of hereditary colorectal cancer and is caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have an increased lifetime risk of developing colorectal cancer as well as other extracolonic tumours. The aim of the current study was to evaluate the frequency and distribution of mutations in the MLH1, MSH2 and MSH6 genes within a cohort of Cypriot families that fulfilled the revised Bethesda guidelines. The study cohort included 77 patients who fulfilled at least one of the revised Bethesda guidelines. Mutational analysis revealed the presence of 4 pathogenic mutations, 3 in the MLH1 gene and 1 in the MSH2 gene, in 5 unrelated individuals. It is noted that out of the 4 pathogenic mutations detected, one is novel (c.1610delG in exon 14 of the MLH1) and has been detected for the first time in the Cypriot population. Overall, the pathogenic mutation detection rate in our patient cohort was 7%. This percentage is relatively low but could be explained by the fact that the sole criterion for genetic screening was compliance to the revised Bethesda guidelines. Larger numbers of Lynch syndrome families and screening of the two additional predisposition genes, PMS2 and EPCAM, are needed in order to decipher the full spectrum of mutations associated with Lynch syndrome predisposition in Cyprus.     

Unique DNA Repair Gene Variations and Potential Associations with the Primary Antibody Deficiency Syndromes IgAD and CVID

Background

Despite considerable effort, the genetic factors responsible for >90% of the antibody deficiency syndromes IgAD and CVID remain elusive. To produce a functionally diverse antibody repertoire B lymphocytes undergo class switch recombination. This process is initiated by AID-catalyzed deamination of cytidine to uridine in switch region DNA. Subsequently, these residues are recognized by the uracil excision enzyme UNG2 or the mismatch repair proteins MutSα (MSH2/MSH6) and MutLα (PMS2/MLH1). Further processing by ubiquitous DNA repair factors is thought to introduce DNA breaks, ultimately leading to class switch recombination and expression of a different antibody isotype.

Methodology/Principal Findings

Defects in AID and UNG2 have been shown to result in the primary immunodeficiency hyper-IgM syndrome, leading us to hypothesize that additional, potentially more subtle, DNA repair gene variations may underlie the clinically related antibody deficiencies syndromes IgAD and CVID. In a survey of twenty-seven candidate DNA metabolism genes, markers in MSH2, RAD50, and RAD52 were associated with IgAD/CVID, prompting further investigation into these pathways. Resequencing identified four rare, non-synonymous alleles associated with IgAD/CVID, two in MLH1, one in RAD50, and one in NBS1. One IgAD patient carried heterozygous non-synonymous mutations in MLH1, MSH2, and NBS1. Functional studies revealed that one of the identified mutations, a premature RAD50 stop codon (Q372X), confers increased sensitivity to ionizing radiation.

Conclusions

Our results are consistent with a class switch recombination model in which AID-catalyzed uridines are processed by multiple DNA repair pathways. Genetic defects in these DNA repair pathways may contribute to IgAD and CVID.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

Promoter Methylation of MLH1, PMS2, MSH2 and p16 Is a Phenomenon of Advanced-Stage HCCs

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.     

Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome characterized by familial predisposition to colorectal carcinoma and extracolonic cancers of the gastrointestinal, urological, and female reproductive tracts. This dominant disorder is caused by germline defects in one of at least five DNA mismatch repair (MMR) genes: hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 (GTBP). Germline mutations of hMSH2 and hMLH1 are also frequently identified in families not fulfilling all the Amsterdam criteria, thereby demonstrating that the involvement of these genes is not confined to typical HNPCC. To evaluate the respective involvement of the various MMR genes in typical and incomplete HNPCC syndromes, we have performed an analysis of the hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in a large series of French kindreds (n=75) with colorectal tumors and/or aggregation of extracolonic cancers belonging to the HNPCC spectrum. Mutational analysis has been performed in all families, without preselection for the tumor phenotype. We have detected 26 pathogenic germline mutations of the hMLH1 and hMSH2 genes and several novel variants of the hPMS1, hPMS2, and hMSH6 genes. Our data confirm that, regardless of the type of families and the tumor phenotype, hPMS1, hPMS2, and hMSH6 germline mutations are rare in familial aggregation of colorectal cancers. Furthermore, they suggest that the presence of multiple primary malignancies in a single individual and the observation of extracolonic tumors in relatives of a colorectal cancer patient should be included among the guidelines for referring patients for genetic testing. Electronic Publication     

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.     

Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1

Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR. One of these mutations, E109K, was reported to abrogate the catalytic activity of the enzyme, yet, in the crystal structure of the EXO1/DNA complex, this glutamate is far away from both DNA and the catalytic site of the enzyme. In an attempt to elucidate the reason underlying the putative loss of function of this variant, we expressed it in Escherichia coli, and tested its activity in a series of biochemical assays. We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates. In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.     

Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes

Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3], [4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins – in addition to their role in DNA-damage recognition – decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.     

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

Regulation of theSaccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle,meiosis and after irradiation

The expression of theSRS2 gene, which encodes a DNA helicase involved in DNA repair inSaccharomyces cerevisiae, was studied using anSRS2-lacZ fusion integrated at the chromosomalSRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression ofSRS2 rises at a time nearly coincident with commitment to recombination. Sincesrs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity to G1 cells or Srs2 functions in a repair mechanism associated with replication.     

Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair

A previously unrecognized mismatch repair activity is described. Extracts of immortalized MSH2-deficient mouse fibroblasts did not correct most single base mispairs. The same extracts carried out efficient repair of A/C mismatches. A/G mispairs were less efficiently corrected and there was no significant repair of A/A. MLH1-defective mouse extracts also repaired an A/C mispair. A/C correction by Msh2(-/-) mouse cell extracts was not affected by antibodies against the PMS2 protein, which inhibited long-patch mismatch repair. A/C repair activity is thus independent of MutSalpha, MutSbeta and MutLalpha. A/C mismatches were corrected 5-fold more efficiently by extracts of Msh2 knockout mouse cells than by comparable extracts prepared from hMSH2- or hMLH1-deficient human cells. MSH2-independent A/C correction by mouse cell extracts did not require a nick in the circular duplex DNA substrate. Repair involved replacement of the A and was associated with the resynthesis of a limited stretch of 相似文献   

The Prevention of Repeat-Associated Deletions in Saccharomyces Cerevisiae by Mismatch Repair Depends on Size and Origin of Deletions

We have investigated the effects of mismatch repair on 1- to 61-bp deletions in the yeast Saccharomyces cerevisiae. The deletions are likely to involve unpaired loop intermediates resulting from DNA polymerase slippage. The mutator effects of mutations in the DNA polymerase δ (POL3) gene and the recombinational repair RAD52 gene were studied in combination with mismatch repair defects. The pol3-t mutation increased up to 1000-fold the rate of extended (7-61 bp) but not of 1-bp deletions. In a rad52 null mutant only the 1-bp deletions were increased (12-fold). The mismatch repair mutations pms1, msh2 and msh3 did not affect 31- and 61-bp deletions in the pol3-t but increased the rates of 7- and 1-bp deletions. We propose that loops less than or equal to seven bases generated during replication are subject to mismatch repair by the PMS1, MSH2, MSH3 system and that it cannot act on loops >=31 bases. In contrast to the pol3-t, the enhancement of 1-bp deletions in a rad52 mutant is not altered by a pms1 mutation. Thus, mismatch repair appears to be specific to errors of DNA synthesis generated during semiconservative replication.     

The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.