The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model

Copyright @ 2012 Elsevier. The article can be accessed from the link below.This article has been made available through the Brunel Open Access Publishing Fund.Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a dynamic GAA repeat expansion mutation within intron 1 of the FXN gene. Studies of mouse models for other trinucleotide repeat (TNR) disorders have revealed an important role of mismatch repair (MMR) proteins in TNR instability. To explore the potential role of MMR proteins on intergenerational GAA repeat instability in FRDA, we have analyzed the transmission of unstable GAA repeat expansions from FXN transgenic mice which have been crossed with mice that are deficient for Msh2, Msh3, Msh6 or Pms2. We find in all cases that absence of parental MMR protein not only maintains transmission of GAA expansions and contractions, but also increases GAA repeat mutability (expansions and/or contractions) in the offspring. This indicates that Msh2, Msh3, Msh6 and Pms2 proteins are not the cause of intergenerational GAA expansions or contractions, but act in their canonical MMR capacity to protect against GAA repeat instability. We further identified differential modes of action for the four MMR proteins. Thus, Msh2 and Msh3 protect against GAA repeat contractions, while Msh6 protects against both GAA repeat expansions and contractions, and Pms2 protects against GAA repeat expansions and also promotes contractions. Furthermore, we detected enhanced occupancy of Msh2 and Msh3 proteins downstream of the FXN expanded GAA repeat, suggesting a model in which Msh2/3 dimers are recruited to this region to repair mismatches that would otherwise produce intergenerational GAA contractions. These findings reveal substantial differences in the intergenerational dynamics of expanded GAA repeat sequences compared with expanded CAG/CTG repeats, where Msh2 and Msh3 are thought to actively promote repeat expansions.This study is funded under European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS. This article is made available through the Brunel Open Access Publishing Fund

Pms2 suppresses large expansions of the (GAA·TTC)n sequence in neuronal tissues

Copyright @ 2012 Bourn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)(n) sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)(n) sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)(n) sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)(n) sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)(n) sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.IDB was supported by a postdoctoral fellowship from the National Ataxia Foundation. RMP was supported by Ataxia UK. SA was supported by The Wellcome Trust. This research was made possible by grants from the National Institutes of Health (NIH/NINDS) and the Muscular Dystrophy Association to S.I.B

Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1

The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-Pms1-independent and Mlh1-Pms1-dependent MMR reactions in vitro and in activation of the Mlh1-Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR

Construction of Fluorescently-Tagged and Adenosine Nucleotide-Binding Mutations of the Human MutS Homolog Heterodimer MSH2-MSH3

DNA mismatch repair (MMR) is a highly conserved system for correcting mispaired nucleotides arising from misincorporation errors during DNA replication, genetic recombination, and chemical or physical damage. The MutS homologues (MSH) and MutL homologues (MLH/PMS) are the fundamental components of MMR and are conserved from bacteria to humans. The MSH proteins initiate MMR via mismatch legion recognition. One human MSH complex in particular, hMSH2-hMSH3, recognizes small insertion deletion loops (IDL) and repetitive DNA sequences. Inherited mutations in many MMR genes including hMSH2 lead to a predisposition for colorectal cancer (hereditary non-polyposis colorectal cancer, HNPCC). Also, the hMSH2-hMSH3 complex has been implicated in the expansion of tri-nucleotide repeats in disorders such as Huntington’s disease and myotonic dystrophy. The role hMSH2-hMSH3 plays in this expansion remains enigmatic. Two mutations made to the Walker A nucleotide binding domain of hMSH2 and hMSH3 will allow for a detailed study of the mechanics of this complex in recognizing and binding DNA lesions, as well as the signaling of downstream MMR components. A detailed study of the conformational changes the protein undergoes in lesion recognition will also be possible via fluorescently tagged MSH2 and MSH3 subunits for use in fluorescence resonance energy transfer.Dean's Undergraduate Research Fund AwardNo embarg

Role of Mismatch Repair Enzymes in GAA•TTC Triplet-repeat Expansion in Friedreich Ataxia Induced Pluripotent Stem Cells

The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA•TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA•TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA•TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA•TTC triplet repeats expanded at a rate of about two GAA•TTC triplet repeats/replication. However, GAA•TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA•TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA•TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA•TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system

Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. Methodology/Principal Findings: We have generated fibroblast cells and neural stem cells (NSCs) from control Y47R mice (9 GAA repeats) and GAA repeat expansion YG8R mice (190+120 GAA repeats). We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs) exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR) gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. Conclusions/Significance: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy, for gene therapy, and as a source of cells for cell therapy testing in FRDA mice. © 2014 Sandi et al

Functional Characterization of MutS Homologue Mismatch Repair Proteins and their Variants

Lynch syndrome (LS) is one of the most common hereditary cancer syndromes and may lead to cancer development, mainly in colon or in endometrium, for 20 years earlier than in general population. LS is an autosomal dominantly inherited disorder, associated with the malfunction of a highly conserved postreplicative DNA mismatch repair (MMR) mechanism and germline mutations at least in four different MMR genes, MLH1, MSH2, MSH6, and PMS2. The MMR genes MSH3 and MLH3 have also been linked to LS but their roles are less clear. To be able to offer an appropriate follow-up and genetic counseling to LS families and their mutation carriers, they must be diagnosed, which usually starts by studying the cancer history in the family and the tumor phenotype of the index patient followed by mutation search and pathogenicity assessment of found variations. The amino acid changes which change only one amino acid in a protein structure, do not necessarily destroy protein and therefore their pathogenicity is difficult to interpret. Furthermore, in rare cases, an individual can carry two variations either in the same or different MMR genes, which further complicates the pathogenicity assessment. DNA MMR corrects mismatches arising mainly during DNA replication. DNA synthesis in each cell division is carried out by three major replicative DNA polymerases (Pols) α, δ and ε and their incomplete proofreading activity together with malfunction in MMR leads to the accumulation of mismatches in the genome leading to genomic instability and cancer. MutS homologue (MSH) MMR proteins form the DNA mismatch recognizing factors MutSα (MSH2/MSH6) and MutSβ (MSH2/MSH3). One aim in the present study was to analyze the substrate efficiencies of MutSα and MutSβ by using the functional in vitro MMR assay with different substrates and cell lines. The target substrates of MutSα and MutSβ have already been widely studied. However, the extent of their functional redundancy and clinical substance remains unclear. Here, our results show that although MutSα alone seems to be responsible for the mismatch and one nucleotide loop repair, MutSα and MutSβ have functional redundancy in two nucleotide loop repair and MutSβ even seems to exceed MutSα in that. The finding is clinically relevant since such a strong role in two nucleotide loop repair indicates MSH3 deficiency in tumors with low dinucleotide and no mononucleotide repeat instability. The second aim in the study was to functionally characterize a possible compound effect of 9 pairs of variants of unknown significance (VUS) found in cancer patients. Four variant pairs were shown to be proficient while one VUS, MSH2 c.380A>G was individually assessed proficient but in a pair with another VUS deficient. Thus, our results suggest that two inherited MMR gene variations in a cancer patient may have a concomitant contribution to MMR deficiency. Moreover, the role of this frequently reported MMR gene VUS MSH2 c.380A>G is especially interesting, since its concomitant defect with another variant could finally explain its recurrent occurrence in colorectal cancer patients. Three MSH6 VUS were shown to cause MMR deficiency individually. Furthermore, one separately studied MSH3 variation was shown to be proficient in MMR. The third aim was to study the role of replicative polymerases α and ε in MMR. Here, we demonstrate a proliferating cell nuclear antigen independent interaction between replicative DNA polymerases and MSH proteins MSH2 and MSH6 by co-purification as well as by conventional and chromatin immunoprecipitation. Chromatin recruitment but not the release of MSH2 appears to depend on DNA replication. The novel interaction provides a potential mechanism for replication-dependent strand discrimination during MMR. In addition, we showed that polymerases of the replication fork have a functional role in human MMR. Our data, suggesting that MSH2 and MSH6 physically interact with Pols δ and Pol α, are in accordance with models where MSH proteins are continuously loaded onto chromatin in a replication-dependent manner and persist on DNA that has already completed replication.Lynchin syndrooma (LS) on yksi yleisimmistä periytyvistä syöpätaudeista ja se altistaa syövän kehittymiselle pääasiassa paksusuoleen tai kohdun limakalvolle. Periytyvä syöpä todetaan noin keskimäärin 20-vuotta aikaisemmin kuin vastaavat sporadiset syövät. LS periytyy autosomaalisesti dominantisti, ja se on yhdistetty konservoituneen DNA-virheitä korjaavan mismatch repair (MMR) -korjausjärjestelmän toimintahäiriöön sekä LS:lle altistaviin ituradan mutaatioihin ainakin neljässä eri MMR geenissä: MLH1, MSH2, MSH6 ja PMS2. Myös MMR geenit MSH3 ja MLH3 on yhdistetty Lynchin syndroomaan, mutta niiden rooli siinä on vielä epäselvä. Lynchin syndroomaperheille ja mutaationkantajille tarjottavan geneettisen neuvonnan ja seurannan edellytyksenä on taudin toteaminen. Diagnosointi aloitetaan tutkimalla suvun syöpähistoriaa ja potilaiden kasvainten ilmiasua, minkä jälkeen mutaatioita etsitään potilailta ja mahdollisesti löytyneen variaation patogeenisuus pyritään arvioimaan. Aminohappomuutokset, jotka muuttavat vain yhden aminohapon proteiinien rakenteessa, eivät välttämättä muuta proteiinin toimintaa merkittävästi, mikä vaikeuttaa variaation patogeenisuuden tulkintaa. Lisäksi harvoissa tapauksissa, joissa yksilö kantaa kahta eri variaatiota, joko samassa tai eri MMR-geenissä, patogeenisuuden arviointi on erityisen haastavaa. MMR-korjausmekanismi korjaa emäspariutumavirheitä, jotka syntyvät DNA:n kahdentumisen yhteydessä. Solun jakautuessa DNA:n synteesi tapahtuu kolmen replikatiivisen polymeraasin (pol) α, δ ja ε toimesta. Polymeraasien epätäydellinen oikoluku yhdistettynä MMR -korjausjärjestelmän toimintahäiriöön, johtaa DNA-virheiden kerääntymiseen genomiin, mikä aiheuttaa genomin epävakautta ja voi johtaa syövän kehittymiseen. MMR-proteiinien MutS-homologit muodostavat emäspariutumavirheitä tunnistavat MutSα (MSH2/MSH6) ja MutSβ (MSH2/MSH3) -heterodimeerikompleksit. Yksi työn tavoitteista oli analysoida MutSα- ja MutSβ-proteiinikompleksien korjaustehokkuutta erityyppisissä emäspariutumavirheissä käyttäen hyväksi toiminnallista in vitro MMR testiä. Vaikka MutSα- ja MutSβ-kompleksien kohteena olevia substraatteja on tutkittu paljon, on niiden rooli korjauksessa ja sen kliininen merkitys on vielä epäselvä. Työn tulokset osoittavat, että MutSα näyttäisi olevan yksinään vastuussa GT-emäsparivirheiden ja yhden nukleotidin silmukkarakenteiden korjauksesta, mutta MutSα ja MutSβ -heterodimeerikomplekseilla on toiminnallinen päällekkäisyys kahden nukleotidin silmukkarakenteen korjauksessa, jossa MutSβ näyttäisi olevan tehokkaampi kun MutSα. Löydös on kliinisesti merkittävä, koska MutSβ:n vahva rooli kahden nukleotidin silmukoiden korjauksessa indikoi MSH3-proteiinin toimimattomuutta kasvaimissa, joissa on vain vähäistä dinukleotiditoistojaksojen epävakautta, mutta vakaat mononukleotiditoistojaksot. Työn toisena tavoitteena oli toiminnallisesti karakterisoida syöpäpotilailta löytyneiden 9 erilaisen variaatioparin mahdollista yhteisvaikutusta. Neljän tutkitun variaatioparin ei todettu vaikuttavan proteiinien korjauskykyyn. MSH2-variaation c.380A>G, joka ei yksinään esiintyessään vaikuttanut korjauskykyyn, havaittiin yhdessä toisen variaation kanssa aiheuttavan tilastollisesti merkitsevän korjauskyvyn aleneman. Työn tulokset viittaavat siihen, että kaksi perittyä MMR-geenivariaatiota syöpäpotilaalla saattavat yhdessä aiheuttaa puutoksen MMR korjauksessa. Lisäksi usein raportoidun MSH2 c.380A>G MMR-geenivariaation rooli on erityisen kiinnostava, koska sen havaittu yhteisvaikutus toisen variaation kanssa saattaa vihdoin selittää sen, miksi sitä tavataan toistuvasti paksusuolisyöpäpotilailla. Kolmessa tutkitusta parista MMR-korjauksen puutteen todettiin johtuvan yksittäisestä patogeenisesta MSH6-variaatiosta. Työssä tutkittu MSH3 geenin variaatio ei vaikuttanut MMR-korjaukseen. Työn kolmas tavoite oli tutkia replikatiivisten polymeraasien α ja ε osuutta MMR-mekanismissa. Työssä osoitettiin, että MSH-proteiinit ovat suorassa vuorovaikutuksessa replikatiiviset polymeraasien kanssa eikä PCNA (proliferating cell nuclear antigen) toimi näiden vuorovaikutusten välittäjänä, kuten aikaisemmin on arveltu. DNA:n replikaatio näyttää vaikuttavan MSH2:n kromatiiniin liittymiseen, mutta ei sen irtoamiseen siitä. Lisäksi työssä osoitettiin, että polymeraaseilla α ja ε on toiminnallinen rooli MMR-korjauksessa. Tulosten perusteella voidaan sanoa, että malli, jossa MSH2 interaktoi fyysisesti pol δ:n kanssa ja MSH6 taas pol α:n kanssa, sopii aiemmin julkaistuun havaintoon, että MSH proteiineja liitetään jatkuvasti kromatiiniin replikaation aikana, mutta ne pysyvät sitoutuneena jo kahdentuneessa DNA:ssa