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1.
Int J Med Sci ; 17(6): 815-823, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218703

RESUMO

Importin-11 (Ipo11) is a novel member of the human importin family of transport receptors (karyopherins), which are known to mediate the nucleocytoplasmic transport of protein and RNA cargos. Despite its role in the transport of protein, we found that knockout of Ipo11 nuclear import factor affects normal embryonic development and govern embryo-lethal phenotypes in mice. In this study, we for the first time produced a mouse line containing null mutation in Ipo11 gene utilized by gene trapping. The Ipo11-/- embryos showed an embryonic lethal phenotype. The Ipo11-/- embryos showed a reduced size at embryonic day 10.5 (E10.5) when compared with Ipo11+/+ or Ipo11+/- embryos and died by E11.5. Whereas Ipo11+/- mice were healthy and fertile, and there was no detectable changes in embryonic lethality and phenotype when reviewed. In the X-gal staining with the Ipo11-/- or Ipo11+/- embryos, strong X-gal staining positivity was detected systematically in the whole mount embryos at E10.5, although almost no X-gal positivity was detected at E9.5, indicating that the embryos die soon after the process of Ipo11 expression started. These results indicate that Ipo11 is essential for the normal embryonic development in mice.

2.
Environ Int ; 137: 105528, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32014791

RESUMO

The "Blue Economy (BE)" is an increasingly popular concept as a strategy for safeguarding the world's oceans and water resources. It may emerge when economic activity is in balance with the long term capacity of ocean ecosystems to support the activity in a sustainable manner. Importantly, the concept of BE posits the inherent conflicts between two discourses-growth and development, and protection of ocean resources. The inherent conflicts require solutions to embrace the opportunities associated with the ocean economy while recognizing and addressing its threats. The potential solutions on a global scale are advocated by the United Nations in their Sustainable Development Goals (SDGs). However, we notice that the identification of the scope and boundaries of the BE in line with the UN's SDGs is vague even challenging, and the key stakeholders and their interests and roles in the BE are also vague. This review examines the scientific evidence of the association between the BE and the UN's SDGs, and relevance and alignment of stakeholders on the link between the BE and SDGs. Based on a literature survey between 1998 and 2018, we find that BE is highly associated with SDGs 14-17. Notably, we find that stakeholders prefer SDG 3 Good Health & Well-Being and SDG 8 Decent Work & Economic Growth in the BE context. As stakeholder involvement shows some differences and variations in the relationship between the BE and SDGs, we consider that stakeholders can play some roles directly or indirectly in the BE-SDGs context. In order to set achievable goals and targets in BE-SDGs, we support that key stakeholders should be identified to play several important roles in prosperous economic, societal development and setting tolerable ranges for the ocean biosphere.

3.
Int J Med Sci ; 16(12): 1557-1563, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839743

RESUMO

E2F3, a member of the E2F family, plays a critical role in cell cycle and proliferation by targeting downstream, retinoblastoma (RB) a tumor suppressor family protein. The purpose of this study, was to investigate the role and function of E2F3 in vivo. We examined phenotypic abnormalities, by deletion of the E2f3 gene in mice. Complete ablation of the E2F3 was fully penetrant, in the pure C57BL/6N background. The E2f3+/ - mouse embryo developed normally without fatal disorder. However, they exhibited reduced body weight, growth retardation, skeletal imperfection, and poor grip strength ability. Findings suggest that E2F3 has a pivotal role in muscle and bone development, and affect normal mouse growth.


Assuntos
Desenvolvimento Ósseo/genética , Fator de Transcrição E2F3/genética , Desenvolvimento Embrionário/genética , Músculo Esquelético/crescimento & desenvolvimento , Animais , Apoptose/genética , Peso Corporal/genética , Ciclo Celular/genética , Proliferação de Células/genética , Embrião de Mamíferos , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Fenótipo
4.
Cell Rep ; 28(4): 1090-1102.e3, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340146

RESUMO

In budding yeast, a single DNA double-strand break (DSB) triggers the activation of Mec1ATR-dependent DNA damage checkpoint. After about 12 h, cells turn off the checkpoint signaling and adapt despite the persistence of the DSB. We report that the adaptation involves the autophosphorylation of Mec1 at site S1964. A non-phosphorylatable mec1-S1964A mutant causes cells to arrest permanently in response to a single DSB without affecting the initial kinase activity of Mec1. Autophosphorylation of S1964 is dependent on Ddc1Rad9 and Dpb11TopBP1, and it correlates with the timing of adaptation. We also report that Mec1's binding partner, Ddc2ATRIP, is an inherently stable protein that is degraded specifically upon DNA damage. Ddc2 is regulated extensively through phosphorylation, which, in turn, regulates the localization of the Mec1-Ddc2 complex to DNA lesions. Taken together, these results suggest that checkpoint response is regulated through the autophosphorylation of Mec1 kinase and through the changes in Ddc2 abundance and phosphorylation.

5.
Genes (Basel) ; 10(4)2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965655

RESUMO

Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência/genética
6.
Biomed Chromatogr ; 33(2): e4388, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30238481

RESUMO

In this study, we developed a method for the determination of Penicillium griseofulvum-oriented pyripyropene A (PPPA), a selective inhibitor of acyl-coenzyme A:cholesterol acyltransferase 2, in mouse and human plasma and validated it using liquid chromatography-tandem mass spectrometry. Pyripyropene A (PPPA) and an internal standard, carbamazepine, were separated using a Xterra MS C18 column with a mixture of acetonitrile and 0.1% formic acid as the mobile phase. The ion transitions monitored in positive-ion mode [M + H]+ of multiple-reaction monitoring (MRM) were m/z 148.0 from m/z 584.0 for PPPA and m/z 194.0 from m/z 237.0 for the internal standard. The detector response was specific and linear for PPPA at concentrations within the range from 1 to 5,000 ng/mL. The intra-/inter-day precision and accuracy of the method was acceptable by the criteria for assay validation. The matrix effects of PPPA ranged from 97.6 to 104.2% and from 93.3 to 105.3% in post-preparative mouse and human plasma samples, respectively. PPPA was also stable under various processing and/or handling conditions. Finally, PPPA concentrations in the mouse plasma samples could be measured after intravenous, intraperitoneal, or oral administration of PPPA, suggesting that the assay is useful for pharmacokinetic studies on mice and applicable to human studies.


Assuntos
Cromatografia Líquida/métodos , Penicillium/química , Piridinas/sangue , Piridinas/farmacocinética , Sesquiterpenos/sangue , Sesquiterpenos/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Estabilidade de Medicamentos , Modelos Lineares , Masculino , Camundongos , Camundongos Endogâmicos ICR , Piridinas/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sesquiterpenos/química , Esterol O-Aciltransferase/antagonistas & inibidores
7.
Exp Biol Med (Maywood) ; 243(5): 408-417, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29409347

RESUMO

FRY like transcription coactivator ( Fryl) gene located on chromosome 5 is a paralog of FRY microtubule binding protein ( Fry) in vertebrates. It encodes a protein with unknown functions. Fryl gene is conserved in various species ranging from eukaryotes to human. Although there are several reports on functions of Fry gene, functions of Fryl gene remain unclear. A mouse line containing null mutation in Fryl gene by gene trapping was produced in this study for the first time. The survival and growth of Fryl-/- mice were observed. Fryl gene expression levels in mouse tissues were determined and histopathologic analyses were conducted. Most Fryl-/- mice died soon after birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. No abnormal histopathologic lesion was apparent in full-term embryo or adult tissues except the kidney. Abnormal lining cell layer detachments from walls of collecting and convoluted tubules in kidneys were apparent in Fryl-/- neonates and full-term embryos. Fryl gene was expressed in renal tubular tissues including the glomeruli and convoluted and collecting tubules. This indicates that defects in tubular systems are associated with Fryl functions and death of Fryl-/- neonates. Fryl protein is required for normal development and functional maintenance of kidney in mice. This is the first report of in vivo Fryl gene functions. Impact statement FRY like transcription coactivator ( Fryl) gene is conserved in various species ranging from eukaryotes to human. It expresses a protein with unknown function. We generated a Fryl gene mutant mouse line and found that most homozygous mice died soon after their birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. Full-term mutant embryos showed abnormal collecting and convoluted tubules in kidneys where Fryl gene was expressed. Collectively, these results indicate that Fryl protein is required for normal development and functional maintenance of kidney in mice. To the best of our knowledge, this is the first report on in vivo Fryl gene functions.


Assuntos
Hidronefrose/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Glomérulos Renais/embriologia , Túbulos Renais/embriologia , Proteínas de Membrana/genética , Animais , Linhagem Celular , Feminino , Hidronefrose/mortalidade , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Glomérulos Renais/patologia , Túbulos Renais/patologia , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Ann Rehabil Med ; 41(1): 104-112, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28289642

RESUMO

OBJECTIVE: To investigate the efficacy of portable microcurrent therapy device (PMTD) of the hip internal rotators in the treatment of in-toeing gait caused by increased femoral anteversion in children over 8 years of age. METHODS: Eleven children (22 legs; 4 boys and 7 girls; mean age, 10.4±1.6 years) with in-toeing gait caused by increased femoral anteversion were included in the present study. All children received 60 minutes of PMTD (intensity, 25 µA; frequency, 8 Hz) applied to the hip internal rotators daily for 4 weeks. Hip internal rotation (IR) angle, external rotation (ER) angle, and midmalleolar-second toe angle (MSTA) measurement during stance phase at transverse plane and Family Satisfaction Questionnaire, frequency of tripping and fatigue like pains about the PMTD were performed before treatment and at 4 weeks after initial PMTD treatment. Paired t-test and Fisher exact test were used for statistical analysis. RESULTS: Hip IR/ER/MSTA was 70.3°±5.4°/20.1°±5.5°/-11.4°±2.7°, and 55.7°±7.8°/33.6°±8.2°/-2.6°±3.8° before treatment and at 4 weeks after initial PMTD treatment, respectively (p<0.01). Ten of 11 (91%) children's family stated that they were generally satisfied with the PMTD treatment. The frequency of tripping and fatigue like pains was significantly lower at 4 weeks after PMTD treatment (p<0.05). Excellent inter-rater and intra-rater reliability was observed for repeated MSTA measurements between the examiners (k=0.91-0.96 and k=0.93-0.99), respectively. CONCLUSION: PMTD of the hip internal rotators can be effective in improving the gait pattern of children with in-toeing gait caused by increased femoral anteversion.

9.
Artigo em Inglês | MEDLINE | ID: mdl-27117731

RESUMO

Koreans consume much seafood; the country is surrounded on the east, west and south by the sea. Koreans have eaten raw sashimi for a long time. However, a concern in the raw sea food industry is that the parasitic nematode Anisakis simplex L3 occurs naturally in marine fish. Thus, the fishery industry needs a non-thermal processing method. High hydrostatic pressure (HPP) has been demonstrated to be effective. White spotted conger flesh containing 20 live larvae was exposed to different pressures (150 and 200 MPa for 1 and 5 min; 250 and 300 MPa each for 1 min). The viability of A. simplex L3 was significantly (p < 0.05) reduced in the flesh of white spotted conger by the stepwise increase of high pressure and time. The conditions required to eliminate A. simplex L3 were as follows: 200 MPa for 5 min or 300 MPa for 1 min. The flesh of the white spotted conger treated at 300 MPa for 1 min was whiter and yellower than untreated controls or that treated at 200 MPa for 5 min. No significant changes (p > 0.05) in any of the Hunter colour ('L', 'a' and 'b') values were found after HPP at 200 MPa for 5 min. The fresh treated at 300 MPa for 1 min scored < 4.0 (the defect limit of quality) of flavour, texture and overall acceptability in untrained sensory evaluation using a seven-point hedonic scale. However, the flesh treated at 200 MPa for 5 min scored > 5.0 ('like') for all sensory parameters. This study suggested that HPP at 200 MPa for 5 min could potentially be used for the inactivation of A. simplex L3 in raw fishery food products without any concomitant changes in their colour or sensory qualities.


Assuntos
Anisakis , Enguias/parasitologia , Manipulação de Alimentos/métodos , Pressão Hidrostática , Alimentos Marinhos/parasitologia , Animais , Anisaquíase/prevenção & controle , Qualidade dos Alimentos , Humanos , Larva , República da Coreia , Sensação
10.
Biofouling ; 32(4): 497-509, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26980068

RESUMO

Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases.


Assuntos
Biofilmes/crescimento & desenvolvimento , Interações Hidrofóbicas e Hidrofílicas , Percepção de Quorum , Vibrio parahaemolyticus , Fímbrias Bacterianas , Contaminação de Alimentos/prevenção & controle , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Humanos , Alimentos Marinhos/microbiologia , Vibrio parahaemolyticus/patogenicidade , Vibrio parahaemolyticus/fisiologia
11.
Int J Food Microbiol ; 211: 73-8, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26184763

RESUMO

In this study, the effects of 10-300 mWs/cm(2) of ultraviolet radiation (UV-C) at 260 nm were investigated for the inactivation of two foodborne viruses: murine norovirus-1 (MNV-1; a human norovirus [NoV] surrogate) and hepatitis A virus (HAV). We used an experimentally contaminated stainless steel surface, a common food-contact surface, to examine the effects of low doses of UV-C radiation on MNV-1 and HAV titers. The modified Gompertz equation was used to generate non-linear survival curves and calculate dR-values as the UV-C dose of 90% reduction for MNV-1 (R(2)=0.95, RMSE=0.038) and HAV (R(2)=0.97, RMSE=0.016). Total MNV-1 and HAV titers significantly decreased (p<0.05) with higher doses of UV-C. MNV-1 and HAV were reduced to 0.0-4.4 and 0.0-2.6 log10PFU/ml, respectively, on the stainless steel surfaces by low-dose UV-C treatment. The dR-value, 33.3 mWs/cm(2) for MNV-1 was significantly (p<0.05) lower than 55.4 mWs/cm(2) of HAV. Therefore, the present study shows that HAV is more resistant to UV-C radiation than MNV-1. These data suggest that low doses of UV-C light on food contact surfaces could be effective to inactivate human NoV and HAV in restaurant, institutional, and industrial kitchens and facilities.


Assuntos
Vírus da Hepatite A/efeitos da radiação , Norovirus/efeitos da radiação , Aço Inoxidável/análise , Esterilização/métodos , Animais , Manipulação de Alimentos/instrumentação , Vírus da Hepatite A/crescimento & desenvolvimento , Humanos , Camundongos , Norovirus/crescimento & desenvolvimento , Células RAW 264.7 , Raios Ultravioleta
12.
Sci Rep ; 4: 7439, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25501038

RESUMO

Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics.


Assuntos
Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , DNA Bacteriano/genética , Gonorreia/diagnóstico , Gonorreia/microbiologia , Humanos , Neisseria gonorrhoeae/genética , Temperatura de Transição
13.
Nat Struct Mol Biol ; 21(1): 103-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24336221

RESUMO

In budding yeast, a single double-strand break (DSB) triggers extensive Tel1 (ATM)- and Mec1 (ATR)-dependent phosphorylation of histone H2A around the DSB, to form γ-H2AX. We describe Mec1- and Tel1-dependent phosphorylation of histone H2B at T129. γ-H2B formation is impaired by γ-H2AX and its binding partner Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL7, GAL10 and GAL1 genes is turned off, γ-H2AX is restored within 5 min, in a Mec1-dependent manner; after reinduction of these genes, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other pericentromeric regions, again depending on Mec1. Our data provide new insights in the function and establishment of phosphorylation events occurring on chromatin after DSB induction.


Assuntos
Dano ao DNA , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosforilação , Saccharomyces cerevisiae/genética , Transcrição Genética
14.
J Nanosci Nanotechnol ; 13(4): 2708-13, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23763148

RESUMO

In this study, the Poly(vinylidene fluoride-trifluoethylene) (PVDF) electrospun fibers were successfully prepared by electrospinning. Processing parameters, such as solvents and solution temperature were varied to study their influence on fiber dimensions. Electrospun PVDF fibers were characterized by scanning electron microscope (SEM), Fourier transform infrared spectrophotometer (FT-IR), wide angle X-ray diffraction (WAXD) and differential scanning calorimetry (DSC). The result indicated that the solvent component and temperature have great influence on fiber dimensions. 19% PVDF dissolved in DMF/MEK mixed solvents with the ratio of 8:2 was considered to be most suitable in this study. Furthermore, the increasing of solution temperature can probably induce the formation of beta-phases in electrospun PVDF Fibers.

15.
J Mater Sci Mater Med ; 24(8): 2029-36, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23661256

RESUMO

In this work, novel poly(ε-caprolactone) (PCL) fibrous membranes incorporating amphiphilic polyhedral oligosilsesquioxane (POSS) telechelic (PEG-POSS telechelic) were prepared via electrospinning. The unique microstructure, morphology, thermal stability of the resulting PCL/PEG-POSS telechelic electrospun nanowebs were investigated by X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis, respectively. The addition of amphiphilic PEG-POSS telechelic strongly influenced the fiber diameters, microstructures of the resultant PCL/PEG-POSS telechelic nanofibers, compared to pure PCL nanofibers. The potential biomedical applications of such PEG-POSS telechelic nanowebs as a scaffolding material were also evaluated in vitro using mouse osteoblast-like MC3T3-E1 cells. The cell adhesion, spreading, and interaction behavior of pure PCL and PCL/PEG-POSS telechelic fibrous membranes were explored. It was found that electrospun PCL fibrous membranes incorporating amphiphilic PEG-POSS telechelic showed higher initial cell attachment than pure PCL due to the higher surface free energy of POSS siloxanes. Moreover, the obtained PCL/PEG-POSS telechelic fibrous scaffolds were found to be nontoxic and to maintain the good adhesion ratio between cells and surface (about ~93 %) after cell culturing for 24 h.


Assuntos
Compostos de Organossilício/química , Osteoblastos/citologia , Poliésteres/química , Polietilenoglicóis/química , Tecidos Suporte , Animais , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/instrumentação , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galvanoplastia/métodos , Camundongos , Compostos de Organossilício/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Poliésteres/síntese química , Polietilenoglicóis/farmacologia , Tensoativos/química , Tensoativos/farmacologia , Tecidos Suporte/química , Difração de Raios X
16.
PLoS Genet ; 8(11): e1003026, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144625

RESUMO

Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s) responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR) events after a DNA double-strand break (DSB) in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Replicação do DNA/genética , Translocação Genética/genética , Aberrações Cromossômicas , Cromossomos/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Genética , Saccharomyces cerevisiae
17.
PLoS Genet ; 8(4): e1002630, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496671

RESUMO

During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB) at MAT is repaired by recombining with one of two donors, HMLα or HMRa, located at opposite ends of chromosome III. MATa cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE), located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MATa cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MATa cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MATa then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX) around the DSB but can also promote γ-H2AX spreading around the RE region.


Assuntos
Proteínas de Ciclo Celular , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead , Genes Fúngicos Tipo Acasalamento , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Bactérias , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases
18.
Nat Struct Mol Biol ; 17(12): 1478-85, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21102445

RESUMO

The repair of DNA double-strand breaks (DSBs) by homologous recombination is essential for genomic stability. The first step in this process is resection of 5' strands to generate 3' single-stranded DNA intermediates. Efficient resection in budding yeast requires the Mre11-Rad50-Xrs2 (MRX) complex and the Sae2 protein, although the role of MRX has been unclear because Mre11 paradoxically has 3'→5' exonuclease activity in vitro. Here we reconstitute resection with purified MRX, Sae2 and Exo1 proteins and show that degradation of the 5' strand is catalyzed by Exo1 yet completely dependent on MRX and Sae2 when Exo1 levels are limiting. This stimulation is mainly caused by cooperative binding of DNA substrates by Exo1, MRX and Sae2. This work establishes the direct role of MRX and Sae2 in promoting the resection of 5' strands in DNA DSB repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Endonucleases/fisiologia , Exodesoxirribonucleases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/química , Endonucleases/química , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/química
19.
Nature ; 454(7203): 543-6, 2008 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-18650924

RESUMO

Chromosome translocations are frequently associated with many types of blood-related cancers and childhood sarcomas. Detection of chromosome translocations assists in diagnosis, treatment and prognosis of these diseases; however, despite their importance to such diseases, the molecular mechanisms leading to chromosome translocations are not well understood. The available evidence indicates a role for non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs) in their origin. Here we develop a yeast-based system that induces a reciprocal chromosome translocation by formation and ligation of breaks on two different chromosomes. We show that interchromosomal end joining is efficiently suppressed by the Tel1- and Mre11-Rad50-Xrs2-dependent pathway; this is distinct from the role of Tel1 in telomeric integrity and from Mec1- and Tel1-dependent checkpoint controls. Suppression of DSB-induced chromosome translocations depends on the kinase activity of Tel1 and Dun1, and the damage-induced phosphorylation of Sae2 and histone H2AX proteins. Tel1- and Sae2-dependent tethering and promotion of 5' to 3' degradation of broken chromosome ends discourage error-prone NHEJ and interchromosomal NHEJ, preserving chromosome integrity on DNA damage. Our results indicate that, like human ATM, Tel1 serves as a key regulator for chromosome integrity in the pathway that reduces the risk for DSB-induced chromosome translocations, and are probably pertinent to the oncogenic chromosome translocations in ATM-deficient cells.


Assuntos
Proteínas de Ciclo Celular , Quebra Cromossômica , Reparo do DNA , Proteínas de Ligação a DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Translocação Genética , Proteínas Supressoras de Tumor , Proteínas Mutadas de Ataxia Telangiectasia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética
20.
Genetics ; 176(4): 2003-14, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17565964

RESUMO

Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches [5-20 nucleotides (nt)] of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single-stranded DNA (ssDNA) via a few bases (<5 nt) or extensive direct repeat homologies (>20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 3' flap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.


Assuntos
DNA Fúngico/genética , DNA Fúngico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Quebras de DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase beta , Reparo do DNA , Enzimas Reparadoras do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genes Fúngicos , Modelos Genéticos , Endonucleases Específicas para DNA e RNA de Cadeia Simples
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