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Estudo do papel da homeostase do cálcio na reparação de danos no DNA em diferentes linhagens de Saccharomyces cerevisiaePoletto, Nadine Paese 21 November 2008 (has links)
O Ca+2 tem sido relatado como um íon de extrema importância celular. Sua concentração intracelular alterada está relacionada com: (i) a ativação de proteínas importantes, dentre elas as de reparação e de apoptose, (ii) as doenças humanas de grande relevância sócio-econômica e (iii) o aumento dos danos celulares. Sabendo-se que a sua concentração intracelular é responsável pela manutenção da viabilidade celular e frente à importância já conhecida deste íon, esta dissertação teve por objetivo, primeiramente, fazer uma busca sobre os dados existentes na literatura para delinear quais são os possíveis mecanismos moleculares pelo qual o Ca+2 afeta os mecanismos de reparação e apoptose. Esta busca norteou posteriores testes e pesquisas que culminaram nos resultados obtidos e apresentados neste trabalho. Para a realização de alguns testes foi desenvolvida uma nova metodologia, o ensaio Poissoner Quantitative Drop Test (PQDT), a qual permite a análise de sobrevivência de diferentes linhagens de Saccharomyces cerevisiae deficientes ou proficientes em determinadas proteínas, frente à exposição crônica a um agente genotóxico. Esta técnica também permite observar efeitos citostáticos e citotóxicos nas células testadas. Além disso, realizou-se uma análise de Biologia de Sistemas das proteínas de S. cerevisiae responsáveis pela homeostase de Ca+2 e encontrou-se uma correlação direta destas com proteínas responsáveis pela reparação de DNA por meio da via de excisão de nucleotídeos ou NER. Uma vez obtidos estes dados, as linhagens de leveduras deficientes nas proteínas tanto de homeostase de Ca+2, Pmr1p, Cod1p, Por1p, como para o gene RAD4 foram construídas. Além dos simples mutantes, foram construídas linhagens duplos mutantes para ambas as vias. Assim, foram realizados ensaios de citotoxicidade, citostaticidade e de parada do ciclo celular frente à exposição ao agente genotóxico 4-NQO. Conforme os resultados obtidos, a homeostase de Ca+2 exerce influência sobre os mecanismos de reparação de DNA, principalmente nas proteínas da via NER, como a Rad4p. As proteínas Pmr1p e Cod1p são as proteínas de homeostase de Ca+2 mais importantes na resposta ao dano celular. Estes resultados propõem que o estresse de retículo endoplasmático e a fosforilação de proteínas de parada de ciclo celular foram os principais processos metabólicos evidenciados nas linhagens de leveduras testadas após a exposição ao agente genotóxico 4-NQO. Os resultados da análise de Biologia de Sistemas e análise ontológica dos genes mostram que existem muitas vias bioquímicas relacionadas com a homeostase de Ca+2. Isso demonstra a importância destes achados e predispõem muitos outros testes para a elucidação completa destas influências. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-22T16:41:02Z
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Dissertacao Nadine Paese Poletto.pdf: 2514929 bytes, checksum: e52f9e75c1db5e89a4fbaf0d3b14215f (MD5) / Made available in DSpace on 2014-05-22T16:41:02Z (GMT). No. of bitstreams: 1
Dissertacao Nadine Paese Poletto.pdf: 2514929 bytes, checksum: e52f9e75c1db5e89a4fbaf0d3b14215f (MD5) / Calcium (Ca2+) has been reported as an important cellular ion. The changes in the intracellular Ca2+ concentrations are related to: (i) the activation of major proteins related to DNA repair and apoptosis; (ii) induction of human diseases; and (iii) increase in cellular damages. Considering that the Ca2+ intracellular concentration is also responsible for the maintenance of cellular viability, the aim of this study was to proceed with a literature data mining to outline the major Ca2+ molecular mechanisms that can affect the cellular activities. This data mining was necessary to define further biological assays described in this work. In this sense, it was developed a new methodology called PDQT, which allows to evaluate the survival of different Saccharomyces cerevisiae strains deficient or proficient for determined proteins in a chronic exposure conditions in the presence of a genotoxic agent. This new method also allows to observe the citotoxic, citostatic, and cell cycle arrests effects induced by a genotoxic agent. Moreover, it was performed a Systems Biology analyses of the major S. cerevisiae Ca2+ homeostasis-associated proteins and it was found a direct association with proteins of the nucleotide excision repair (NER) pathway. The data gathered by Systems Biology analyses were used to construct yeast strains single and double mutants deficient for Ca2+ homeostasisassociated proteins, like Pmr1p, Cod1p, Por1p, and for the NER-associated protein Rad4p. The yeast strains obtained were challenged against the genotoxic agent 4-NQO and citotoxic, citostatic, and cell cycle arrest assays were performed. Taking into account the results, it was observed that Ca2+ homeostasis mechanisms affect the DNA repair pathways, specially the NER proteins like Rad4p. In this sense, both Cod1p and Pmr1p are the most important Ca2+ homeostasis proteins for the damage cellular response. In addition, these results propose that ER stress and the phosphorylation of cell cycle arrest-associated proteins can be the major metabolic processes after 4-NQO exposure. The results obtained by System Biology and gene ontology analyses proved that many biochemical pathways are related to Ca2+ homeostasis, which demonstrate the importance of the results obtained in this work and lead to other studies.
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Estudo do papel da homeostase do cálcio na reparação de danos no DNA em diferentes linhagens de Saccharomyces cerevisiaePoletto, Nadine Paese 21 November 2008 (has links)
O Ca+2 tem sido relatado como um íon de extrema importância celular. Sua concentração intracelular alterada está relacionada com: (i) a ativação de proteínas importantes, dentre elas as de reparação e de apoptose, (ii) as doenças humanas de grande relevância sócio-econômica e (iii) o aumento dos danos celulares. Sabendo-se que a sua concentração intracelular é responsável pela manutenção da viabilidade celular e frente à importância já conhecida deste íon, esta dissertação teve por objetivo, primeiramente, fazer uma busca sobre os dados existentes na literatura para delinear quais são os possíveis mecanismos moleculares pelo qual o Ca+2 afeta os mecanismos de reparação e apoptose. Esta busca norteou posteriores testes e pesquisas que culminaram nos resultados obtidos e apresentados neste trabalho. Para a realização de alguns testes foi desenvolvida uma nova metodologia, o ensaio Poissoner Quantitative Drop Test (PQDT), a qual permite a análise de sobrevivência de diferentes linhagens de Saccharomyces cerevisiae deficientes ou proficientes em determinadas proteínas, frente à exposição crônica a um agente genotóxico. Esta técnica também permite observar efeitos citostáticos e citotóxicos nas células testadas. Além disso, realizou-se uma análise de Biologia de Sistemas das proteínas de S. cerevisiae responsáveis pela homeostase de Ca+2 e encontrou-se uma correlação direta destas com proteínas responsáveis pela reparação de DNA por meio da via de excisão de nucleotídeos ou NER. Uma vez obtidos estes dados, as linhagens de leveduras deficientes nas proteínas tanto de homeostase de Ca+2, Pmr1p, Cod1p, Por1p, como para o gene RAD4 foram construídas. Além dos simples mutantes, foram construídas linhagens duplos mutantes para ambas as vias. Assim, foram realizados ensaios de citotoxicidade, citostaticidade e de parada do ciclo celular frente à exposição ao agente genotóxico 4-NQO. Conforme os resultados obtidos, a homeostase de Ca+2 exerce influência sobre os mecanismos de reparação de DNA, principalmente nas proteínas da via NER, como a Rad4p. As proteínas Pmr1p e Cod1p são as proteínas de homeostase de Ca+2 mais importantes na resposta ao dano celular. Estes resultados propõem que o estresse de retículo endoplasmático e a fosforilação de proteínas de parada de ciclo celular foram os principais processos metabólicos evidenciados nas linhagens de leveduras testadas após a exposição ao agente genotóxico 4-NQO. Os resultados da análise de Biologia de Sistemas e análise ontológica dos genes mostram que existem muitas vias bioquímicas relacionadas com a homeostase de Ca+2. Isso demonstra a importância destes achados e predispõem muitos outros testes para a elucidação completa destas influências. / Calcium (Ca2+) has been reported as an important cellular ion. The changes in the intracellular Ca2+ concentrations are related to: (i) the activation of major proteins related to DNA repair and apoptosis; (ii) induction of human diseases; and (iii) increase in cellular damages. Considering that the Ca2+ intracellular concentration is also responsible for the maintenance of cellular viability, the aim of this study was to proceed with a literature data mining to outline the major Ca2+ molecular mechanisms that can affect the cellular activities. This data mining was necessary to define further biological assays described in this work. In this sense, it was developed a new methodology called PDQT, which allows to evaluate the survival of different Saccharomyces cerevisiae strains deficient or proficient for determined proteins in a chronic exposure conditions in the presence of a genotoxic agent. This new method also allows to observe the citotoxic, citostatic, and cell cycle arrests effects induced by a genotoxic agent. Moreover, it was performed a Systems Biology analyses of the major S. cerevisiae Ca2+ homeostasis-associated proteins and it was found a direct association with proteins of the nucleotide excision repair (NER) pathway. The data gathered by Systems Biology analyses were used to construct yeast strains single and double mutants deficient for Ca2+ homeostasisassociated proteins, like Pmr1p, Cod1p, Por1p, and for the NER-associated protein Rad4p. The yeast strains obtained were challenged against the genotoxic agent 4-NQO and citotoxic, citostatic, and cell cycle arrest assays were performed. Taking into account the results, it was observed that Ca2+ homeostasis mechanisms affect the DNA repair pathways, specially the NER proteins like Rad4p. In this sense, both Cod1p and Pmr1p are the most important Ca2+ homeostasis proteins for the damage cellular response. In addition, these results propose that ER stress and the phosphorylation of cell cycle arrest-associated proteins can be the major metabolic processes after 4-NQO exposure. The results obtained by System Biology and gene ontology analyses proved that many biochemical pathways are related to Ca2+ homeostasis, which demonstrate the importance of the results obtained in this work and lead to other studies.
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Deletion of the RNR4 gene causes hyperresistance to the carcinogen 4-NQO in the yeast modelBulet, Lisa 08 1900 (has links)
La stabilité génomique, qui est essentielle à la vie, est possible grâce à la réplication et la réparation de l’ADN. Une des enzymes responsables de la réplication et de la réparation de l’ADN est la ribonucleotide reductase (RNR), qui est retrouvée chez la levure et chez l’humain. Cette enzyme catalyse la formation de déoxyribonucléotides et maintien le pool de dNTP requis pour la réparation et la réplication de l’ADN. L’enzyme RNR est un tétramère α2β2 constitué d’une grande (R1, α2) et d’une petite (R2, β2) sous-unité. Chez S. cerevisiae, les gènes RNR1 et RNR3 encodent la sous-unité α2 (R1). L’activité catalytique de RNR dépend d’une interaction avec le fer et de la formation d’un complexe entre R1 et R2. L’expression de toutes les sous-unités est inductible par les dommages causés à l’ADN.
Dans cette étude, nous démontrons que des cellules qui n’expriment pas une des sous-unités, Rnr4, du complexe RNR sont sensibles à divers agents endommageant l’ADN, tels que le méthyl méthane sulfonate, la bléomycine, le péroxyde d’hydrogène et les rayons ultraviolets (UVC 254 nm). Au contraire, le mutant est résistant au 4-nitroquinoline-1- oxide (4-NQO), un composé qui engendre des lésions encombrantes. Par conséquent, le mutant rnr4Δ démontre une réduction marquée en mutations induites par le 4-NQO comparativement à la souche parentale. Nous voulions identifier la voie de réparation de l’ADN qui conférait cette résistance au 4-NQO ainsi que les protéines impliquées. Les voies BER, NER et MMR n’ont pas aboli la résistance au 4-NQO de la souche rnr4Δ. La protéine recombinante Rad51 ne joue pas un rôle critique dans la réparation de l’ADN et dans la résistance au 4-NQO. La délétion du gène REV3, qui encode une polymérase de contournement, impliquée dans la réparation post-réplication, a partiellement aboli la résistance au 4-NQO dans rnr4Δ. Ces résultats suggèrent que la polymérase Rev3 et possiblement d’autres polymérases translésion (Rev1, Rev7, Rad30) pourraient être impliquées dans la réparation de lésions encombrantes dans l’ADN dans des conditions de carence en dNTP.
La réparation de l’ADN, un mécanisme complexe chez la levure, implique une vaste gamme de protéines, dont certaines encore inconnues. Nos résultats indiquent qu’il y aurait plus qu’une protéine impliquée dans la résistance au 4-NQO. Des investigations plus approfondies seront nécessaires afin de comprendre la recombinaison et la réparation post-réplication. / Genomic stability, critical for life, is controlled by DNA replication and repair. DNA replication and repair is mediated through many enzymes, one being ribonucleotide reductase (RNR), an enzyme found in both yeast and humans. RNR catalyzes the reaction involved in the formation of deoxyribonucleotides and is responsible for maintaining dNTP pools required for DNA repair and replication. RNR is an α2β2 tetramer consisting of a large (R1, α2) and small subunit (R2, β2). In S. cerevisiae RNR1 and RNR3 encode α2 (R1). RNR catalytic activity depends on its interaction with iron and on the formation of the complex between R1 and R2. All subunits are inducible by DNA damage.
Here we show that cells lacking one subunit, Rnr4, of the RNR complex are sensitive to various DNA damaging agents such as methyl methane sulfonate, bleomycin, hydrogen peroxide, and ultraviolet radiation (UVC 254 nm). In contrast, the mutant is resistant to 4-nitroquinoline-1-oxide (4-NQO), an agent which produces bulky lesions. Consistent with this resistance, the rnr4Δ showed a sharp reduction in 4-NQO-induced mutations as compared to the parent.
We wanted to determine which pathway was able to confer resistance to 4-NQO and thus targeted DNA repair proteins. The repair pathways BER, NER and MMR did not abolish 4-NQO resistance in rnr4Δ. Recombination protein Rad51 (NHEJ) was lethal in an rnr4Δ thus indicating no role in DNA repair and 4-NQO resistance. Deletion of the REV3 gene, encoding a DNA bypass polymerase involved in post replication repair, partially abolished 4-NQO resistance in rnr4Δ. These results suggest that Rev3 and possibly other translesion polymerases (Rev1, Rev7, Rad30) could play a role in the repair of bulky DNA lesions under low levels of dNTPs.
DNA repair, a complex mechanism in yeast, involves a vast array of proteins, some yet to be discovered. Our results indicate that there is more than one protein involved in 4-NQO resistance and further investigation is required concerning recombination and post replication repair.
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Deletion of the RNR4 gene causes hyperresistance to the carcinogen 4-NQO in the yeast modelBulet, Lisa 08 1900 (has links)
La stabilité génomique, qui est essentielle à la vie, est possible grâce à la réplication et la réparation de l’ADN. Une des enzymes responsables de la réplication et de la réparation de l’ADN est la ribonucleotide reductase (RNR), qui est retrouvée chez la levure et chez l’humain. Cette enzyme catalyse la formation de déoxyribonucléotides et maintien le pool de dNTP requis pour la réparation et la réplication de l’ADN. L’enzyme RNR est un tétramère α2β2 constitué d’une grande (R1, α2) et d’une petite (R2, β2) sous-unité. Chez S. cerevisiae, les gènes RNR1 et RNR3 encodent la sous-unité α2 (R1). L’activité catalytique de RNR dépend d’une interaction avec le fer et de la formation d’un complexe entre R1 et R2. L’expression de toutes les sous-unités est inductible par les dommages causés à l’ADN.
Dans cette étude, nous démontrons que des cellules qui n’expriment pas une des sous-unités, Rnr4, du complexe RNR sont sensibles à divers agents endommageant l’ADN, tels que le méthyl méthane sulfonate, la bléomycine, le péroxyde d’hydrogène et les rayons ultraviolets (UVC 254 nm). Au contraire, le mutant est résistant au 4-nitroquinoline-1- oxide (4-NQO), un composé qui engendre des lésions encombrantes. Par conséquent, le mutant rnr4Δ démontre une réduction marquée en mutations induites par le 4-NQO comparativement à la souche parentale. Nous voulions identifier la voie de réparation de l’ADN qui conférait cette résistance au 4-NQO ainsi que les protéines impliquées. Les voies BER, NER et MMR n’ont pas aboli la résistance au 4-NQO de la souche rnr4Δ. La protéine recombinante Rad51 ne joue pas un rôle critique dans la réparation de l’ADN et dans la résistance au 4-NQO. La délétion du gène REV3, qui encode une polymérase de contournement, impliquée dans la réparation post-réplication, a partiellement aboli la résistance au 4-NQO dans rnr4Δ. Ces résultats suggèrent que la polymérase Rev3 et possiblement d’autres polymérases translésion (Rev1, Rev7, Rad30) pourraient être impliquées dans la réparation de lésions encombrantes dans l’ADN dans des conditions de carence en dNTP.
La réparation de l’ADN, un mécanisme complexe chez la levure, implique une vaste gamme de protéines, dont certaines encore inconnues. Nos résultats indiquent qu’il y aurait plus qu’une protéine impliquée dans la résistance au 4-NQO. Des investigations plus approfondies seront nécessaires afin de comprendre la recombinaison et la réparation post-réplication. / Genomic stability, critical for life, is controlled by DNA replication and repair. DNA replication and repair is mediated through many enzymes, one being ribonucleotide reductase (RNR), an enzyme found in both yeast and humans. RNR catalyzes the reaction involved in the formation of deoxyribonucleotides and is responsible for maintaining dNTP pools required for DNA repair and replication. RNR is an α2β2 tetramer consisting of a large (R1, α2) and small subunit (R2, β2). In S. cerevisiae RNR1 and RNR3 encode α2 (R1). RNR catalytic activity depends on its interaction with iron and on the formation of the complex between R1 and R2. All subunits are inducible by DNA damage.
Here we show that cells lacking one subunit, Rnr4, of the RNR complex are sensitive to various DNA damaging agents such as methyl methane sulfonate, bleomycin, hydrogen peroxide, and ultraviolet radiation (UVC 254 nm). In contrast, the mutant is resistant to 4-nitroquinoline-1-oxide (4-NQO), an agent which produces bulky lesions. Consistent with this resistance, the rnr4Δ showed a sharp reduction in 4-NQO-induced mutations as compared to the parent.
We wanted to determine which pathway was able to confer resistance to 4-NQO and thus targeted DNA repair proteins. The repair pathways BER, NER and MMR did not abolish 4-NQO resistance in rnr4Δ. Recombination protein Rad51 (NHEJ) was lethal in an rnr4Δ thus indicating no role in DNA repair and 4-NQO resistance. Deletion of the REV3 gene, encoding a DNA bypass polymerase involved in post replication repair, partially abolished 4-NQO resistance in rnr4Δ. These results suggest that Rev3 and possibly other translesion polymerases (Rev1, Rev7, Rad30) could play a role in the repair of bulky DNA lesions under low levels of dNTPs.
DNA repair, a complex mechanism in yeast, involves a vast array of proteins, some yet to be discovered. Our results indicate that there is more than one protein involved in 4-NQO resistance and further investigation is required concerning recombination and post replication repair.
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The function of the Saccharomyces Cerevisiae ribonucleotide reductase second [beta] subunit in DNA repairZhao, Chunyu January 2006 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Untersuchungen zur Rolle des <i>MPH1</i>-Gens aus <i>Saccharomyces cerevisiae</i> bei der Reinitiation der Replikation nach schadensinduzierten Arresten / Investigations on the function of the <i>Saccharomyces cerevisiae MPH1</i> gene in reinitation of replication after damage induced arrestsRudolph, Christian 05 November 2003 (has links)
No description available.
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Untersuchungen zur Funktion der Gene MPH1 und MMS2 aus Saccharomyces cerevisiae bei der fehlerfreien Umgehung von replikationsarretierenden DNA-Schäden / Studies on functions of the genes MPH1 and MMS2 from Saccharomyces cerevisiae during error free bypass of replication blocking DNA-lesionsEde, Christopher 13 January 2010 (has links)
No description available.
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Histone modifications after DNA damage affect survival in Schizosaccharomyces pombe / Rajput, Abdul Mateen January 2010 (has links)
S. cerevisiae Ada2 and Bre1 has a role in histone post-translational modifications. Deletion of these genes causes deficiency in acetylation (Ada2) or ubiquitination (Bre1) of histones. Further, mutants lacking these genes or homologous genes showed different phenotypes in human and S. cerevisiae while treated with DNA damaging agents 4-NQO and MMS. Bre1 deficient cells showed 4-NQO sensitivity in S. cerevisiae and resistance in human cells. Since it has been shown that S. pombe is more close to mammals in chromatin regulation we wanted to examine S. pombe response against MMS and 4-NQO. By homologous recombination, genes were deleted and mutants were treated with different concentration of both the genotoxins. In accordance with a previous study, Ada2Δ showed sensitivity to MMS while Brl1Δ & Brl2Δ grew as wild type. Surprisingly, unlike S. cerevisiae, S. pombe showed resistance to 4-NQO and has a phenotype similar to the one found in human cells.
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