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.     

Phosphorylation of mismatch repair proteins MSH2 and MSH6 affecting MutSalpha mismatch-binding activity

Mismatch repair (MMR) is involved in the removal of mispaired bases from DNA and thus plays an important role in the maintenance of genomic stability and the prevention of mutations and cancer. Moreover, MMR triggers genotoxicity and apoptosis upon processing of DNA lesions such as O⁶-methylguanine. Whereas the enzymology of MMR has been elucidated in great detail, only limited data are available concerning its regulation. Here we show that the major mismatch-binding proteins MSH2 and MSH6, forming the MutSα complex, are phosphorylated in vitro by protein kinase C and casein kinase II, but not by protein kinase A. Phosphorylation of MSH2 and MSH6 was also found within the cell, with MSH6 being more extensively phosphorylated than MSH2. Lack of MSH2 and MSH6 phosphorylation in vivo due to phosphate depletion, kinase inhibition (by H7 and quercetin) and treatment with phosphatases (CIP, SAP and λ-PPase) significantly reduced mismatch-binding activity of MutSα. It also prevented methylation-induced nuclear translocation of the repair complex, indicating that nuclear translocation of MutSα upon mutagen treatment is dependent on protein phosphorylation. The finding that MSH2 and MSH6 are subject to phosphorylation resulting in increased mismatch binding by MutSα indicates a novel type of post-translational regulation of MMR which might be involved in the response of cells to genotoxic stress.     

Dissimilar mispair-recognition spectra of Arabidopsis DNA-mismatch-repair proteins MSH2*MSH6 (MutSalpha) and MSH2*MSH7 (MutSgamma)

Besides orthologs of other eukaryotic mismatch-repair (MMR) proteins, plants encode MSH7, a paralog of MSH6. The Arabidopsis thaliana recognition heterodimers AtMSH2·MSH6 (AtMutSα) and AtMSH2·MSH3 (AtMutSβ) were previously found to bind the same subsets of mismatches as their counterparts in other eukaryotes—respectively, base–base mismatches and single extra nucleotides, loopouts of extra nucleotides (one or more) only—but AtMSH2·MSH7 (AtMutSγ) bound well only to a G/T mismatch. To test hypotheses that MSH7 might be specialized for G/T, or for base mismatches in 5-methylcytosine contexts, we compared binding of AtMutSα and AtMutSγ to a series of mismatched DNA oligoduplexes, relative to their (roughly similar) binding to G/T DNA. AtMutSγ bound G/G, G/A, A/A and especially C/A mispairs as well or better than G/T, in contrast to MutSα, for which G/T was clearly the best base mismatch. The presence of 5-methylcytosine adjacent to or in a mispair generally lowered binding by both heterodimers, with no systematic difference between the two. Alignment of protein sequences reveals the absence in MSH7 of the clamp domains that in bacterial MutS proteins—and by inference MSH6 proteins—non-specifically bind the backbone of mismatched DNA, raising new questions as to how clamp domains enhance mismatch recogni tion. Plants must rigorously suppress mutation during mitotic division of meristematic cells that eventually give rise to gametes and may also use MMR proteins to antagonize homeologous recombination. The MSH6 versus MSH7 divergence may reflect specializations for particular mismatches and/or sequence contexts, so as to increase both DNA-replication and meiotic-recombination fidelity, or dedication of MSH6 to the former and MSH7 to the latter, consistent with genetic evidence from wheat.     

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process.     

Transfer of the MSH2.MSH6 complex from proliferating cell nuclear antigen to mispaired bases in DNA

Proliferating cell nuclear antigen (PCNA) is thought to play a role in DNA mismatch repair at the DNA synthesis step as well as in an earlier step. Studies showing that PCNA interacts with mispair-binding protein complexes, MSH2.MSH3 and MSH2.MSH6, and that PCNA enhances MSH2.MSH6 mispair binding specificity suggest PCNA may be involved in mispair recognition. Here we show that PCNA and MSH2.MSH6 form a stable ternary complex with a homoduplex (G/C) DNA, but MSH2.MSH6 binding to a heteroduplex (G/T) DNA disrupts MSH2.MSH6 binding to PCNA. We also found that the addition of ATP or adenosine 5'-O-(thiotriphosphate) restores MSH2.MSH6 binding to PCNA, presumably by disrupting MSH2.MSH6 binding to the heteroduplex (G/T) DNA. These results support a model in which MSH2.MSH6 binds to PCNA loaded on newly replicated DNA and is transferred from PCNA to mispaired bases in DNA.     

MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae.

Oxidation of G in DNA yields 8-oxo-G (GO), a mutagenic lesion that leads to misincorporation of A opposite GO. In E. coli, GO in GO:C base pairs is removed by MutM, and A in GO:A mispairs is removed by MutY. In S. cerevisiae, mutations in MSH2 or MSH6 caused a synergistic increase in mutation rate in combination with mutations in OGG1, which encodes a MutM homolog, resulting in a 140- to 218-fold increase in the G:C-to-T:A transversion rate. Consistent with this, MSH2-MSH6 complex bound to GO:A mispairs and GO:C base pairs with high affinity and specificity. These data indicate that in S. cerevisiae, MSH2-MSH6-dependent mismatch repair is the major mechanism by which misincorporation of A opposite GO is corrected.     

Functional interaction of proliferating cell nuclear antigen with MSH2-MSH6 and MSH2-MSH3 complexes

Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs.     

EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches. In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease. Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees. DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions. The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively. Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay. High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality. These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.     

Human MSH6 deficiency is associated with impaired antibody maturation

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.     

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.     

Molecular cloning of zebrafish (Danio rerio) MutS homolog 6(MSH6) and noncoordinate expression of MSH6 gene activity and G-T mismatch binding proteins in zebrafish larvae

Eukaryotic MutS homolog 6(MSH6) is a DNA mismatch recognition protein associated with mismatch repair of simple base-base mispairs and small insertion-deletion loops. As replication or recombination errors generated during embryonic development of living organisms should be efficiently corrected to maintain the integrity of genetic materials, we attempted to study MSH6 gene expression in developing zebrafish (Danio rerio) and the influence of MSH6 expression on the production of mismatch binding factors. A full-length cDNA encoding zebrafish MSH6 (zMSH6) was first obtained by rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of zMSH6 shares 57% and 56% identity with human and mouse MSH6, respectively. The 190-kDa recombinant zMSH6 containing 1,369 amino acids bound preferentially to a heteroduplex than to a homoduplex DNA. Northern blot and semiquantitative RT-PCR analysis detected apparent levels of zMSH6 mRNA expression in 12 and 36-hr-old zebrafish embryos, while this expression in 84-hr-old larvae was dramatically reduced to 23% of that in 12-hr-old embryos when beta-actin mRNA was constitutively synthesized. Incubation of G-T and G-G heteroduplex probes with 12 to 60-hr-old zebrafish extracts produced predominantly high-shifting binding complexes with very similar band intensity. Although low in zMSH6 mRNA production, the extracts of 84-hr-old larvae interacted significantly stronger than the embryonic extracts with both G-T and G-G mispairs, producing high and low-shifting complexes. Heteroduplex-recognition proteins in 108-hr-old larvae gave a similar pattern of mismatch binding. The intensities of G-T complexes produced by 84 and 108-hr-old zebrafish extracts were 2.5 to 3-fold higher than those of G-G complexes. Our data indicate that the production of efficient MSH6-independent binding factors, particularly G-T-specific recognition proteins, is upregulated in zebrafish at the larval stage when MSH6 gene activity is downregulated.     

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.     

Up-regulation of MSH receptors by MSH in Cloudman melanoma cells.

MSH can up-regulate MSH binding capacity of cultured Cloudman melanoma cells in a dose- and time-dependent fashion. Binding is mediated through proteins exhibiting an apparent molecular weight of 50-53kDa, consistent with previous studies implicating them as the principal MSH receptors on Cloudman cells. Pre-incubation of cells with MSH stimulates expression of the receptor proteins both on the plasma membrane surface as well as in internal sites associated with coated vesicles. The effects of MSH are additive with those of UV light, suggesting that UV and MSH might stimulate receptor expression through separate mechanisms.     

Conformational Change in MSH2-MSH6 upon Binding DNA Coupled to ATPase Activity

Postreplication DNA mismatch repair is initiated by the eukaryotic protein MSH2-MSH6 or the prokaryotic protein MutS, both showing overall conserved structure and functionality. Crystal structures of MSH2-MSH6 and MutS bound to the mismatch DNA reveal a closed architecture of the clamp and the lever domains exhibiting strong contacts with the bent DNA backbone. Long molecular dynamics simulations of the human MSH2-MSH6 protein in the absence of a DNA show an altered conformation of the protein that reflects the protein's state before binding to DNA. The clamp and the lever domains of both MSH6 and MSH2 open in an asymmetric and dramatic fashion. The opening of the clamp and the lever domains in the absence of DNA is coupled to changes in the ATPase domains, which explains the experimentally observed diminished ATPase activity in DNA-free MSH2-MSH6 and illustrates the allosteric coupling between DNA binding and ATPase activity.     

Induction of two DNA mismatch repair proteins, MSH2 and MSH6, in differentiated human neuroblastoma SH-SY5Y cells exposed to doxorubicin

The MutS homologues MSH2 and MSH6 form a heterodimeric protein complex that is involved in the recognition of base/base mismatches and insertion/deletion loops, as well as some other types of DNA damage. We investigated the expression of these proteins in undifferentiated and retinoic acid-differentiated human neuroblastoma SH-SY5Y cells by immunocytochemistry, western blot analysis, and RT-PCR. Nuclei from undifferentiated SH-SY5Y cells were found to be immunoreactive to anti-MSH2 and anti-MSH6 antibodies. Following differentiation, the cells stop dividing and change morphology to acquire a neuron-like phenotype. Under these conditions, both anti-MSH2 and anti-MSH6 immunoreactivities were still detectable, although the signals were somewhat less intense. When these cells were exposed for 2 h to neurotoxic concentrations of doxorubicin (50 nM), they exhibited a marked and homogeneous increase of both anti-MSH2 and anti-MSH6 immunoreactivities. As revealed by western blot analysis, these effects were associated with increased protein content and were dose-dependent. Using RT-PCR technology, we also found that doxorubicin treatment did not change MSH2 or MSH6 mRNA levels. Our data indicate that human postmitotic, neuron-like cells constitutively express the molecular machinery devoted to recognition of DNA mismatches and that this system is activated by specific treatment leading to cell death. These findings might help clarify the molecular mechanisms underlying various human neurological diseases that are associated with deficiencies in DNA repair and/or a high rate of DNA damage acquisition.     

Deciphering the Mismatch Recognition Cycle in MutS and MSH2-MSH6 Using Normal-Mode Analysis

Postreplication DNA mismatch repair is essential for maintaining the integrity of genomic information in prokaryotes and eukaryotes. The first step in mismatch repair is the recognition of base-base mismatches and insertions/deletions by bacterial MutS or eukaryotic MSH2-MSH6. Crystal structures of both proteins bound to mismatch DNA reveal a similar molecular architecture but provide limited insight into the detailed molecular mechanism of long-range allostery involved in mismatch recognition and repair initiation. This study describes normal-mode calculations of MutS and MSH2-MSH6 with and without DNA. The results reveal similar protein flexibilities and suggest common dynamic and functional characteristics. A strongly correlated motion is present between the lever domain and ATPase domains, which suggests a pathway for long-range allostery from the N-terminal DNA binding domain to the C-terminal ATPase domains, as indicated by experimental studies. A detailed analysis of individual low-frequency modes of both MutS and MSH2-MSH6 shows changes in the DNA-binding domains coupled to the ATPase sites, which are interpreted in the context of experimental data to arrive at a complete molecular-level mismatch recognition cycle. Distinct conformational states are proposed for DNA scanning, mismatch recognition, repair initiation, and sliding along DNA after mismatch recognition. Hypotheses based on the results presented here form the basis for further experimental and computational studies.     

MSH2/MSH6 Complex Promotes Error-Free Repair of AID-Induced dU:G Mispairs as well as Error-Prone Hypermutation of A:T Sites

Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2^−/−Msh6^−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice are indistinguishable from Msh2^−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2^−/−, Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2^−/−Msh6^−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM.     

Penetrance and expressivity of MSH6 germline mutations in seven kindreds not ascertained by family history

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by inherited mutations in DNA mismatch-repair genes, most commonly MLH1 or MSH2. The role MSH6 plays in inherited cancer susceptibility is less well defined. The aim of this study was to investigate the penetrance and expressivity of MSH6 mutations in kindreds ascertained through endometrial cancer probands unselected for family history. Detailed pedigrees were constructed for six MSH6 mutation carriers. All reported cancers and precancers were confirmed, and tissues were obtained when available. Tumors were analyzed for microsatellite instability (MSI) and for expression of MSH2, MLH1, and MSH6. MSH6 mutation status was determined for 59 family members. Of these 59 individuals, 19 (32%) had confirmed cancers and precancers. There was an excess of mutation carriers among the 19 affected family members (11 [58%] of 19) compared with those among the 40 unaffecteds (8 [20%] of 40, P=.0065, odds ratio = 5.5, 95% CI = 1.66-18.19). In four of the seven tumors analyzed from mutation carriers other than the probands, MSI and/or MMR protein expression was consistent with the involvement of MSH6. Overall estimated penetrance of the MHS6 mutations was 57.7%. Of the tumors in mutation carriers, 78% were part of the extended HNPCC spectrum. This study demonstrates that MSH6 germline mutations are, indeed, associated with increased cancer risk and that the penetrance of mutations may be higher than appreciated elsewhere. A combination of MSI and immunohistochemistry analyses may be helpful in screening for MSH6 mutation carriers.