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.     

Functional analysis of HNPCC-related missense mutations in MSH2

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.     

A Genetic Incompatibility Accelerates Adaptation in Yeast

During mismatch repair (MMR) MSH proteins bind to mismatches that form as the result of DNA replication errors and recruit MLH factors such as Mlh1-Pms1 to initiate excision and repair steps. Previously, we identified a negative epistatic interaction involving naturally occurring polymorphisms in the MLH1 and PMS1 genes of baker’s yeast. Here we hypothesize that a mutagenic state resulting from this negative epistatic interaction increases the likelihood of obtaining beneficial mutations that can promote adaptation to stress conditions. We tested this by stressing yeast strains bearing mutagenic (incompatible) and non-mutagenic (compatible) mismatch repair genotypes. Our data show that incompatible populations adapted more rapidly and without an apparent fitness cost to high salt stress. The fitness advantage of incompatible populations was rapid but disappeared over time. The fitness gains in both compatible and incompatible strains were due primarily to mutations in PMR1 that appeared earlier in incompatible evolving populations. These data demonstrate a rapid and reversible role (by mating) for genetic incompatibilities in accelerating adaptation in eukaryotes. They also provide an approach to link experimental studies to observational population genomics.     

Characterization of components of the mismatch repair machinery in Trypanosoma brucei

Mismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base-base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.     

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.     

Identification of a Comprehensive Spectrum of Genetic Factors for Hereditary Breast Cancer in a Chinese Population by Next-Generation Sequencing

The genetic etiology of hereditary breast cancer has not been fully elucidated. Although germline mutations of high-penetrance genes such as BRCA1/2 are implicated in development of hereditary breast cancers, at least half of all breast cancer families are not linked to these genes. To identify a comprehensive spectrum of genetic factors for hereditary breast cancer in a Chinese population, we performed an analysis of germline mutations in 2,165 coding exons of 152 genes associated with hereditary cancer using next-generation sequencing (NGS) in 99 breast cancer patients from families of cancer patients regardless of cancer types. Forty-two deleterious germline mutations were identified in 21 genes of 34 patients, including 18 (18.2%) BRCA1 or BRCA2 mutations, 3 (3%) TP53 mutations, 5 (5.1%) DNA mismatch repair gene mutations, 1 (1%) CDH1 mutation, 6 (6.1%) Fanconi anemia pathway gene mutations, and 9 (9.1%) mutations in other genes. Of seven patients who carried mutations in more than one gene, 4 were BRCA1/2 mutation carriers, and their average onset age was much younger than patients with only BRCA1/2 mutations. Almost all identified high-penetrance gene mutations in those families fulfill the typical phenotypes of hereditary cancer syndromes listed in the National Comprehensive Cancer Network (NCCN) guidelines, except two TP53 and three mismatch repair gene mutations. Furthermore, functional studies of MSH3 germline mutations confirmed the association between MSH3 mutation and tumorigenesis, and segregation analysis suggested antagonism between BRCA1 and MSH3. We also identified a lot of low-penetrance gene mutations. Although the clinical significance of those newly identified low-penetrance gene mutations has not been fully appreciated yet, these new findings do provide valuable epidemiological information for the future studies. Together, these findings highlight the importance of genetic testing based on NCCN guidelines and a multi-gene analysis using NGS may be a supplement to traditional genetic counseling.     

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.     

A mutation in the MSH6 subunit of the Saccharomyces cerevisiae MSH2-MSH6 complex disrupts mismatch recognition.

In yeast, MSH2 interacts with MSH6 to repair base pair mismatches and single nucleotide insertion/deletion mismatches and with MSH3 to recognize small loop insertion/deletion mismatches. We identified a msh6 mutation (msh6-F337A) that when overexpressed in wild type strains conferred a defect in both MSH2-MSH6- and MSH2-MSH3-dependent mismatch repair pathways. Genetic analysis suggested that this phenotype was due to msh6-F337A sequestering MSH2 and preventing it from interacting with MSH3 and MSH6. In UV cross-linking, filter binding, and gel retardation assays, the MSH2-msh6-F337A complex displayed a mismatch recognition defect. These observations, in conjunction with ATPase and dissociation rate analysis, suggested that MSH2-msh6-F337A formed an unproductive complex that was unable to stably bind to mismatch DNA.     

Mutator phenotypes of common polymorphisms and missense mutations in MSH2.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in the DNA mismatch repair gene hMSH2 [1], the human homologue of the Escherichia coli MutS gene. These are mostly nonsense, frameshift or deletion mutations that result in loss of intact protein and complete inactivation of DNA mismatch repair. However, cancer is also associated with hMSH2 missense mutations that are merely inferred to be deleterious because they result in non-conservative substitutions of amino acids that are highly conserved among MutS family proteins. Moreover, sequence polymorphisms exist in hMSH2 that also change conserved amino acids but whose functional consequences and relationship to cancer are uncertain. Here, we show that yeast strains harboring putative equivalents of three hMSH2 polymorphisms have elevated mutation rates. Mutator effects were also observed for yeast equivalents of hMSH2 missense mutations found in HNPCC families and in an early onset colon tumor. Several distinct phenotypes were observed, indicating that these missense mutations have differential effects on MSH2 function(s). The results suggest that cancer may be associated with even partial loss of hMSH2 function and they are consistent with the hypothesis that polymorphisms in hMSH2 might predispose humans to disease.     

Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells

DNA mismatch repair enzymes (for example, MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of children (～11%) with newly diagnosed acute lymphoblastic leukemia have low or undetectable MSH2 protein levels, despite abundant wild-type MSH2 mRNA. Leukemia cells with low levels of MSH2 contained partial or complete somatic deletions of one to four genes that regulate MSH2 degradation (FRAP1 (also known as MTOR), HERC1, PRKCZ and PIK3C2B); we also found these deletions in individuals with adult acute lymphoblastic leukemia (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2 degradation, leading to substantial reduction in DNA mismatch repair and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, DNA mismatch repair deficiency and drug resistance.     

MSH2 missense mutations alter cisplatin cytotoxicity and promote cisplatin-induced genome instability

Defects in the mismatch repair protein MSH2 cause tolerance to DNA damage. We report how cancer-derived and polymorphic MSH2 missense mutations affect cisplatin cytotoxicity. The chemotolerance phenotype was compared with the mutator phenotype in a yeast model system. MSH2 missense mutations display a strikingly different effect on cell death and genome instability. A mutator phenotype does not predict chemotolerance or vice versa. MSH2 mutations that were identified in tumors (Y109C) or as genetic variations (L402F) promote tolerance to cisplatin, but leave the initial mutation rate of cells unaltered. A secondary increase in the mutation rate is observed after cisplatin exposure in these strains. The mutation spectrum of cisplatin-resistant mutators identifies persistent cisplatin adduction as the cause for this acquired genome instability. Our results demonstrate that MSH2 missense mutations that were identified in tumors or as polymorphic variations can cause increased cisplatin tolerance independent of an initial mutator phenotype. Cisplatin exposure promotes drug-induced genome instability. From a mechanistical standpoint, these data demonstrate functional separation between MSH2-dependent cisplatin cytotoxicity and repair. From a clinical standpoint, these data provide valuable information on the consequences of point mutations for the success of chemotherapy and the risk for secondary carcinogenesis.     

Mismatch recognition function of Arabidopsis thaliana MutSγ

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2–MSH6 and MSH2–MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can^r mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSβ, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.     

Mismatch repair gene mutations in Chinese HNPCC patients

To explore the characteristics of DNA mismatch repair gene mutations in Chinese patients with hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome, the MLH1 and MSH2 genes from probands of 76 HNPCC families were sequenced. By doing so, two frame-shift mutations, three splice-site mutations and fourteen missense mutations (thirteen missense mutations and one nonsense mutation) were identified in the MLH1 gene. In addition, one splice-site mutation and six missense mutations were detected in the MSH2 gene. None of these mutations were detected in 100 matched healthy controls. The remaining mutation-negative cases were subjected to large fragment deletion analysis using multiplex ligation-dependent probe amplification (MLPA). By doing so, five large fragment deletions were detected in the MSH2 gene. No large fragment deletions were detected in the MLH1 gene. We conclude that the MLH1 and MSH2 genes in Chinese HNPCC families exhibit broad mutation spectra.     

DNA repair gene polymorphisms and risk of early onset colorectal cancer in Lynch syndrome

DNA repair plays a pivotal role in maintaining genomic integrity with over 130 genes involved in various repair pathways that include base excision repair, nucleotide excision repair, double strand break repair and DNA mismatch repair. Polymorphisms within genes that are involved in these processes have been widely reported to be associated with cancer susceptibility in an extensive range of malignancies that include colorectal cancer (CRC). Lynch syndrome is caused by inherited germline mutations in DNA mismatch repair genes, predominantly in MLH1 and MSH2, that predispose to a variety of epithelial malignancies, most notably CRC. Despite being a relatively well understood hereditary cancer syndrome there remain several questions in relation to genetic influences on disease expression. Since Lynch syndrome is associated with a breakdown in DNA mismatch repair variation in other DNA repair genes may influence disease expression. In this report we have genotyped 424 Australian and Polish Lynch syndrome participants for eight common DNA repair gene polymorphisms to assess any association with the age of CRC onset. The DNA repair gene SNPs included in the study were: BRCA2 (rs11571653), MSH3 (rs26279), Lig4 (rs1805386), OGG1 (rs1052133), XRCC1 (rs25487), XRCC2 (rs3218536 and rs1799793) and XRCC3 (rs861539). Cox multi-variant regression modelling failed to provide any convincing evidence of an effect in any of the polymorphisms analysed. The data suggest that polymorphisms in DNA repair genes do not contribute to cancer risk in a population of CRC patients who are at increased risk of disease as a result in a deficiency of DNA mismatch repair.     

Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.

Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect. We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae. In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines. These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene. Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides. Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.     

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.     

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.     

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.