RESUMO
The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSα-dependent and MutSß-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level in Saccharomyces cerevisiae. We found that inactivation of MutSα-dependent MMR increases the genome-wide mutation rate by â¼17-fold and loss of MutSß-dependent MMR elevates the genome-wide mutation rate by â¼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSß-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSα-dependent MMR is more important than MutSß-dependent MMR for protection from 1-bp insertions, while MutSß-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5'-NCN-3' trinucleotides, 5'-GCA-3' trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the -1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSß-dependent MMR pathways.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismoRESUMO
Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD). We show MutL-DNA binding is undetectable in physiological conditions. Instead, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone, likely enabling diffusion-mediated wrapping of the remaining MutL domains around the DNA. The resulting MutL sliding clamp enhances MutH endonuclease and UvrD helicase activities on the DNA, which also engage the PCC during strand-specific incision/excision. These MutS clamp-loader progressions are significantly different from the replication clamp-loaders that attach the polymerase processivity factors ß-clamp/PCNA to DNA, highlighting the breadth of mechanisms for stably linking crucial genome maintenance proteins onto DNA.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Reparo do DNA , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Nucleotídeos , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.
Assuntos
Proteínas de Escherichia coli , Proteína MutS de Ligação de DNA com Erro de Pareamento , Humanos , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , DNA/metabolismo , Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas MutS/genética , Ligação ProteicaRESUMO
MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.
Assuntos
Proteínas de Escherichia coli , Proteína MutS de Ligação de DNA com Erro de Pareamento , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/química , DNA/metabolismo , Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismoRESUMO
STUDY QUESTION: Do bi-allelic variants in the genes encoding the MSH4/MSH5 heterodimer cause male infertility? SUMMARY ANSWER: We detected biallelic, (likely) pathogenic variants in MSH5 (4 men) and MSH4 (3 men) in six azoospermic men, demonstrating that genetic variants in these genes are a relevant cause of male infertility. WHAT IS KNOWN ALREADY: MSH4 and MSH5 form a heterodimer, which is required for prophase of meiosis I. One variant in MSH5 and two variants in MSH4 have been described as causal for premature ovarian insufficiency (POI) in a total of five women, resulting in infertility. Recently, pathogenic variants in MSH4 have been reported in infertile men. So far, no pathogenic variants in MSH5 had been described in males. STUDY DESIGN, SIZE, DURATION: We utilized exome data from 1305 men included in the Male Reproductive Genomics (MERGE) study, including 90 males with meiotic arrest (MeiA). Independently, exome sequencing was performed in a man with MeiA from a large consanguineous family. PARTICIPANTS/MATERIALS, SETTING, METHODS: Assuming an autosomal-recessive mode of inheritance, we screened the exome data for rare, biallelic coding variants in MSH4 and MSH5. If possible, segregation analysis in the patients' families was performed. The functional consequences of identified loss-of-function (LoF) variants in MSH5 were studied using heterologous expression of the MSH5 protein in HEK293T cells. The point of arrest during meiosis was determined by γH2AX staining. MAIN RESULTS AND THE ROLE OF CHANCE: We report for the first time (likely) pathogenic, homozygous variants in MSH5 causing infertility in 2 out of 90 men with MeiA and overall in 4 out of 902 azoospermic men. Additionally, we detected biallelic variants in MSH4 in two men with MeiA and in the sister of one proband with POI. γH2AX staining revealed an arrest in early prophase of meiosis I in individuals with pathogenic MSH4 or MSH5 variants. Heterologous in vitro expression of the detected LoF variants in MSH5 showed that the variant p.(Ala620GlnTer9) resulted in MSH5 protein truncation and the variant p.(Ser26GlnfsTer42) resulted in a complete loss of MSH5. LARGE SCALE DATA: All variants have been submitted to ClinVar (SCV001468891-SCV001468896 and SCV001591030) and can also be accessed in the Male Fertility Gene Atlas (MFGA). LIMITATIONS, REASONS FOR CAUTION: By selecting for variants in MSH4 and MSH5, we were able to determine the cause of infertility in six men and one woman, leaving most of the examined individuals without a causal diagnosis. WIDER IMPLICATIONS OF THE FINDINGS: Our findings have diagnostic value by increasing the number of genes associated with non-obstructive azoospermia with high clinical validity. The analysis of such genes has prognostic consequences for assessing whether men with azoospermia would benefit from a testicular biopsy. We also provide further evidence that MeiA in men and POI in women share the same genetic causes. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation sponsored Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326), and supported by institutional funding of the Research Institute Amsterdam Reproduction and Development and funds from the LucaBella Foundation. The authors declare no conflict of interest.
Assuntos
Azoospermia , Infertilidade Masculina , Azoospermia/genética , Proteínas de Ciclo Celular/genética , Reparo de Erro de Pareamento de DNA , Feminino , Células HEK293 , Humanos , Infertilidade Masculina/genética , Masculino , Meiose/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genéticaRESUMO
MutS is a key component of the mismatch repair (MMR) pathway. Members of the MutS protein family are present in prokaryotes, eukaryotes, and viruses. Six MutS homologs (MSH1-6) have been identified in yeast, of which three function in nuclear MMR, while MSH1 functions in mitochondrial DNA repair. MSH proteins are believed to be well conserved in animals, except for MSH1-which is thought to be lost. Two intriguing exceptions to this general picture have been found, both in the class Anthozoa within the phylum Cnidaria. First, an ortholog of the yeast-MSH1 was reported in one hexacoral species. Second, a MutS homolog (mtMutS) has been found in the mitochondrial genome of all octocorals. To understand the origin and potential functional implications of these exceptions, we investigated the evolution of the MutS family both in Cnidaria and in animals in general. Our study confirmed the acquisition of octocoral mtMutS by horizontal gene transfer from a giant virus. Surprisingly, we identified MSH1 in all hexacorals and several sponges and placozoans. By contrast, MSH1 orthologs were lacking in other cnidarians, ctenophores, and bilaterian animals. Furthermore, while we identified MSH2 and MSH6 in nearly all animals, MSH4, MSH5, and, especially, MSH3 were missing in multiple species. Overall, our analysis revealed a dynamic evolution of the MutS family in animals, with multiple losses of MSH1, MSH3, some losses of MSH4 and MSH5, and a gain of the octocoral mtMutS. We propose that octocoral mtMutS functionally replaced MSH1 that was present in the common ancestor of Anthozoa.
Assuntos
Proteínas de Saccharomyces cerevisiae , Animais , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS-MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein-protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5' to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5' â 3' excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.
Assuntos
Pareamento Incorreto de Bases , Reparo de Erro de Pareamento de DNA , Adenosina Trifosfatases , DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genéticaRESUMO
Mismatch repair is a highly conserved cellular pathway responsible for repairing mismatched dsDNA. Errors are detected by the MutS enzyme, which most likely senses altered mechanical property of damaged dsDNA rather than a specific molecular pattern. While the curved shape of dsDNA in crystallographic MutS/DNA structures suggests the role of DNA bending, the theoretical support is not fully convincing. Here, we present a computational study focused on a base-pair opening into the minor groove, a specific base-pair motion observed upon interaction with MutS. Propensities for the opening were evaluated in terms of two base-pair parameters: Opening and Shear. We tested all possible base pairs in anti/anti, anti/syn and syn/anti orientations and found clear discrimination between mismatches and canonical base-pairs only for the opening into the minor groove. Besides, the discrimination gap was also confirmed in hotspot and coldspot sequences, indicating that the opening could play a more significant role in the mismatch recognition than previously recognized. Our findings can be helpful for a better understanding of sequence-dependent mutability. Further, detailed structural characterization of mismatches can serve for designing anti-cancer drugs targeting mismatched base pairs.
Assuntos
Pareamento Incorreto de Bases , Reparo de Erro de Pareamento de DNA , DNA/química , Simulação de Dinâmica Molecular , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pareamento de Bases , Biologia Computacional , DNA/metabolismo , Ligação de Hidrogênio , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , TermodinâmicaRESUMO
Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.
Assuntos
Achromobacter/genética , Fibrose Cística/microbiologia , Taxa de Mutação , Mutação , Achromobacter/efeitos dos fármacos , Antibacterianos/farmacologia , Doença Crônica , Reparo de Erro de Pareamento de DNA , Dinamarca , Humanos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Fenótipo , Prevalência , Rifampina/farmacologiaRESUMO
MutL homolog 1 (MLH1) is a key DNA mismatch repair protein, which plays an important role in maintenance of genomic stability and the DNA damage response. Here, we report that MLH1 is a novel substrate of histone deacetylase 6 (HDAC6). HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLα complex. Moreover, we have identified four novel acetylation sites in MLH1 by MS analysis. The deacetylation mimetic mutant, but not the WT and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSα-MutLα complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSα-MutLα complex by deacetylation of MLH1, leading to the tolerance of DNA damage.
Assuntos
Dano ao DNA , Desacetilase 6 de Histona/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Acetilação/efeitos dos fármacos , Linhagem Celular , Desacetilase 6 de Histona/genética , Humanos , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutação , Tioguanina/farmacologiaRESUMO
MutS homologs identify base-pairing errors made in DNA during replication and initiate their repair. In the presence of adenosine triphosphate, MutS induces DNA bending upon mismatch recognition and subsequently undergoes conformational transitions that promote its interaction with MutL to signal repair. In the absence of MutL, these transitions lead to formation of a MutS mobile clamp that can move along the DNA. Previous single-molecule FRET (smFRET) studies characterized the dynamics of MutS DNA-binding domains during these transitions. Here, we use protein-DNA and DNA-DNA smFRET to monitor DNA conformational changes, and we use kinetic analyses to correlate DNA and protein conformational changes to one another and to the steps on the pathway to mobile clamp formation. The results reveal multiple sequential structural changes in both MutS and DNA, and they suggest that DNA dynamics play a critical role in the formation of the MutS mobile clamp. Taking these findings together with data from our previous studies, we propose a unified model of coordinated MutS and DNA conformational changes wherein initiation of mismatch repair is governed by a balance of DNA bending/unbending energetics and MutS conformational changes coupled to its nucleotide binding properties.
Assuntos
Pareamento Incorreto de Bases/genética , Reparo de Erro de Pareamento de DNA , DNA/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Conformação de Ácido Nucleico , Pareamento de Bases/fisiologia , Reparo de Erro de Pareamento de DNA/genética , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , Instabilidade Genômica/genética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica/fisiologia , Conformação Proteica , Domínios Proteicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS-DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS-DNA complex.
Assuntos
Proteínas de Bactérias/metabolismo , Pareamento Incorreto de Bases , Reparo de Erro de Pareamento de DNA , DNA/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , DNA/química , DNA/genética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização ProteicaRESUMO
DNA mismatch repair (MMR) is necessary to prevent incorporation of polymerase errors into the newly synthesized DNA strand, as they would be mutagenic. In humans, errors in MMR cause a predisposition to cancer, called Lynch syndrome. The MMR process is performed by a set of ATPases that transmit, validate, and couple information to identify which DNA strand requires repair. To understand the individual steps in the repair process, it is useful to be able to study these large molecular machines structurally and functionally. However, the steps and states are highly transient; therefore, the methods to capture and enrich them are essential. Here, we describe how single-cysteine variants can be used for specific cross-linking and labeling approaches that allow trapping of relevant transient states. Analysis of these defined states in functional and structural studies is instrumental to elucidate the molecular mechanism of this important DNA MMR process.
Assuntos
Reagentes de Ligações Cruzadas/química , Cisteína/química , Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli/química , Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutação Puntual , Conformação ProteicaRESUMO
The first Spanish multi-centre study on the microbiology of cystic fibrosis (CF) was conducted from 2013 to 2014. The study involved 24 CF units from 17 hospitals, and recruited 341 patients. The aim of this study was to characterise Pseudomonas aeruginosa isolates, 79 of which were recovered from 75 (22%) patients. The study determined the population structure, antibiotic susceptibility profile and genetic background of the strains. Fifty-five percent of the isolates were multi-drug-resistant, and 16% were extensively-drug-resistant. Defective mutS and mutL genes were observed in mutator isolates (15.2%). Considerable genetic diversity was observed by pulsed-field gel electrophoresis (70 patterns) and multi-locus sequence typing (72 sequence types). International epidemic clones were not detected. Fifty-one new and 14 previously described array tube (AT) genotypes were detected by AT technology. This study found a genetically unrelated and highly diverse CF P. aeruginosa population in Spain, not represented by the epidemic clones widely distributed across Europe, with multiple combinations of virulence factors and high antimicrobial resistance rates (except for colistin).
Assuntos
Fibrose Cística/complicações , Farmacorresistência Bacteriana , Variação Genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Adolescente , Adulto , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Proteínas MutL/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Espanha/epidemiologia , Virulência , Adulto JovemRESUMO
The fragile X-related disorders result from expansion of a CGG/CCG microsatellite in the 5' UTR of the FMR1 gene. We have previously demonstrated that the MSH2/MSH3 complex, MutSß, that is important for mismatch repair, is essential for almost all expansions in a mouse model of these disorders. Here we show that the MSH2/MSH6 complex, MutSα also contributes to the production of both germ line and somatic expansions as evidenced by the reduction in the number of expansions observed in Msh6-/- mice. This effect is not mediated via an indirect effect of the loss of MSH6 on the level of MSH3. However, since MutSß is required for 98% of germ line expansions and almost all somatic ones, MutSα is apparently not able to efficiently substitute for MutSß in the expansion process. Using purified human proteins we demonstrate that MutSα, like MutSß, binds to substrates with loop-outs of the repeats and increases the thermal stability of the structures that they form. We also show that MutSα facilitates binding of MutSß to these loop-outs. These data suggest possible models for the contribution of MutSα to repeat expansion. In addition, we show that unlike MutSß, MutSα may also act to protect against repeat contractions in the Fmr1 gene.
Assuntos
Proteínas de Ligação a DNA/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Mutação , Oligonucleotídeos/genética , Proteínas/genéticaRESUMO
In order to understand how MutS recognizes mismatched DNA and induces the reaction of DNA repair using ATP, the dynamics of the complexes of MutS (bound to the ADP and ATP nucleotides, or not) and DNA (with mismatched and matched base-pairs) were investigated using molecular dynamics simulations. As for DNA, the structure of the base-pairs of the homoduplex DNA which interacted with the DNA recognition site of MutS was intermittently disturbed, indicating that the homoduplex DNA was unstable. As for MutS, the disordered loops in the ATPase domains, which are considered to be necessary for the induction of DNA repair, were close to (away from) the nucleotide-binding sites in the ATPase domains when the nucleotides were (not) bound to MutS. This indicates that the ATPase domains changed their structural stability upon ATP binding using the disordered loop. Conformational analysis by principal component analysis showed that the nucleotide binding changed modes which have structurally solid ATPase domains and the large bending motion of the DNA from higher to lower frequencies. In the MutS-mismatched DNA complex bound to two nucleotides, the bending motion of the DNA at low frequency modes may play a role in triggering the formation of the sliding clamp for the following DNA-repair reaction step. Moreover, MM-PBSA/GBSA showed that the MutS-homoduplex DNA complex bound to two nucleotides was unstable because of the unfavorable interactions between MutS and DNA. This would trigger the ATP hydrolysis or separation of MutS and DNA to continue searching for mismatch base-pairs. Proteins 2016; 84:1287-1303. © 2016 Wiley Periodicals, Inc.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , Pareamento de Bases , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Simulação de Dinâmica Molecular , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Motivos de Nucleotídeos , Análise de Componente Principal , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. RESULTS: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited ~2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by ~3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by ~10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. CONCLUSIONS: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Helicobacter pylori/enzimologia , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Proteínas de Bactérias/genética , Helicobacter pylori/química , Helicobacter pylori/genética , Hidrólise , Cinética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Recombinação GenéticaRESUMO
DNA mismatch repair (MMR) is responsible for correcting errors formed during DNA replication. DNA polymerase errors include base mismatches and extra helical nucleotides referred to as insertion and deletion loops. In bacteria, MMR increases the fidelity of the chromosomal DNA replication pathway approximately 100-fold. MMR defects in bacteria reduce replication fidelity and have the potential to affect fitness. In mammals, MMR defects are characterized by an increase in mutation rate and by microsatellite instability. In this review, we discuss current advances in understanding how MMR functions in bacteria lacking the MutH and Dam methylase-dependent MMR pathway.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Bactérias Gram-Positivas/genética , Adenosina Trifosfatases/metabolismo , Animais , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Aptidão Genética , Bactérias Gram-Positivas/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , MutaçãoRESUMO
During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/ß to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.
Assuntos
DNA Polimerase II/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA/biossíntese , Proteína 2 Homóloga a MutS/genética , Proteínas de Saccharomyces cerevisiae/genética , DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Mutação , Saccharomyces cerevisiaeRESUMO
Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5' to 3' and the 3' to 5' directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3-bp insertion-deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin-distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication.