A specific feature of ionising radiation is the formation of clustered DNA damage, where two or more lesions form within one to two helical turns of the DNA induced by a single radiation track. The complexity of ionising radiation-induced DNA damage increases with increasing ionisation density and it has been shown that complex DNA damage has reduced efficiency of repairability. In mammalian cells, base excision repair (BER) is the predominant pathway for the repair of non-DSB clustered DNA lesions and is split into two sub-pathways known as short patch (SP) BER and long patch (LP) BER. SP-BER is the predominant pathway, especially in the repair of isolated DNA lesions. However, LP-BER is thought to play a greater role in the repair of radiation-induced clustered lesions. In this study, cell lines were generated stably expressing the fluorescently tagged BER proteins, XRCC1-YFP (marker for SP-BER) or FEN1-GFP (marker for LP-BER). The recruitment and loss of XRCC1-YFP and FEN1-GFP to sites of DNA damage induced by both ultrasoft X-ray (USX), a form of low linear energy transfer (LET) radiation, and near infrared (NIR) laser microbeam irradiation (‘mimic’ high LET radiation) was visualised in real-time and the decay kinetics of the fluorescently-tagged proteins determined. The half-life of fluorescence decay of FEN1-GFP following USX irradiation was longer than that of XRCC1-YFP, indicating that LP-BER is a slower process than SP-BER. Additionally, the fluorescence decay of XRCC1-YFP after NIR laser microbeam irradiation was fitted by bi-exponential decays with a fast component and a slow component, reflecting the involvement of XRCC1 in the repair of different types of DNA damage. In contrast to USX irradiation, where the XRCC1-YFP fluorescence decay reached background levels by 20 min, XRCC1-YFP still persisted at some of the NIR laser induced DNA damage sites even after 4 hours. This is consistent with the fact that the laser induces more complex damage that presents a major challenge to the repair proteins, persisting for much longer than the simple damage caused by low LET USX irradiation. Persistent, unrepaired DNA damage can potentially lead to mutations and replication-induced DSBs if it persists into S-phase. PARP1 inhibition reduced the recruitment of XRCC1 to DNA damage sites. However, a considerable amount of XRCC1 was still detected at the DNA damage sites, leading to the conclusion that there is a subset of DNA damage that requires XRCC1 but not PARP1 for repair. Understanding how clustered damage is repaired by the BER pathway can aid the design of future therapies which can be used in combination with radiotherapy to enhance the radiosensitisation effect. Knockdown of FEN1 was investigated and found to radiosensitise A549 (adenocarcinoma) cells, possibly as a result of an excess of unrepaired radiation-induced lesions requiring LP-BER for repair, although FEN1 knockdown alone induced cell death in non-cancerous BEAS-2B cells.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair factor involved in the DNA base excision repair (BER) pathway that is required for the maintenance of genome stability. In this pathway, APE1 cleaves DNA at an abasic site to generate a DNA single strand break, allowing for repair completion by a DNA polymerase and a DNA ligase. High levels of APE1 have been observed in multiple cancer types however it is not understood if this contributes to cancer onset and development. What is known is that these cancers tend to display increased resistance to DNA damaging treatments and APE1 is therefore considered a key target for inhibition in the treatment of APE1-overexpressing cancers. Considering the relevance of modulating APE1 levels in disease and cancer treatment, very little is known about how cellular APE1 levels are regulated. Our lab has previously shown that the levels of the BER factors Pol β, XRCC1 and DNA Lig IIIα are regulated by ubiquitylation-mediated proteasomal degradation. The aim of this doctoral thesis was therefore to determine if ubiquitylation also regulates APE1 stability in cells. I present evidence that APE1 is ubiquitylated in cells and have identified the UBR3 E3 ligase that is responsible for this activity. Using mouse embryonic fibroblasts generated from Ubr3 knockout mice, I demonstrate that UBR3 regulates APE1 cellular levels. I furthermore show that a loss of cellular UBR3 leads to the formation of DNA double strand breaks and genome instability.

A proteína APE1 (do inglêsApurinic/Apyrimidinicendonuclease 1/ Redox Factor-1 - APE1/REF-1) é uma enzima multifuncional, cuja expressão encontra-se frequentemente aumentada em gliomas. Além de apresentar atividade no reparo por excisão de base (BER), o gene APE1 também atua como fator de redução, mantendo fatores de transcrição (FTs) em um estado reduzido ativo. A via BER de reparo do DNA tem sido apontada como um possível fator de resistência a terapias baseadas no uso de agentes alquilantes, tais como temozolomida (TMZ). No presente trabalho, utilizou-se a estratégia de inibição da transcrição do gene APE1 pelo método de RNA interferente(siRNA) e tratamento com a droga TMZ nas células de glioblastoma (GBM), T98G (resistente à TMZ) e U87MG (sensível à TMZ), a fim de verificar a influência do silenciamento do gene APE1 sobre as respostas celulares à droga avaliadas por vários ensaios, bem como os efeitos sobre a expressão transcricional dos genes alvos dos FTs regulados por APE1. O silenciamento de APE1 e o tratamento das células T98G com a TMZ foram eficazes no sentido de reduzir a proliferação e a capacidade clonogênica, além de intervir na progressão do ciclo celular com bloqueio na fase S. Tais efeitos foram acompanhados pelo aumento da indução de danos no DNA e da expressão de H2AX fosforilada (H2AX), o que justifica a queda na sobrevivência celular. O mesmo efeito não foi observado nas células U87MG silenciadas para APE1 e tratadas com a TMZ, havendo o predomínio dos efeitos causados pela TMZ, exceto por uma leve indução de danos no DNA e de H2AX. Adicionalmente, nas células T98G silenciadas e tratadas, verificou-se uma moderada indução de apoptose, que foi observada ao longo dos tempos avaliados (1 a 10 dias), com uma leve indução de caspase-3 (5 dias); nessas células, observou-se também a indução (3,8 vezes) de morte celular autofágica (5 dias). Entretanto, nas células U87MG,a indução de apoptose foi baixa e não houve indução de morte por autofagia, sugerindo outros mecanismos de morte envolvidos na eliminação dessas células em resposta ao tratamento com a TMZ. O silenciamento de APE1 causou uma redução acentuada na invasão das células T98G, de forma similar à observada nas células somente tratadas com a TMZ, sendo que a combinação (silenciamento de APE1 e tratamento com a droga) resultou em um efeito aditivo, enquanto que nas células U87MG a combinação foi eficaz no sentido de reduzir a proporção de células invasivas, fato não observado nas condições isoladas. Os genes COX2 e VEGF, alvos dos FT NFB e HIF-1 (regulados por APE1) foram reprimidos nas células T98G enquanto que o gene VEGF foi induzido nas células U87MG, entretanto, tais alterações no padrão de expressão transcricional foram observadas somente em resposta ao tratamento com a TMZ, independentemente do silenciamento de APE1, indicando nenhuma mudança na atividade redox de APE1, possivelmente pela existência de proteínas APE1 remanescentes na célula. Além disso, a expressão proteica de NFBp65(ser563) foi aumentada em ambas as linhagens silenciadas e tratadas com a TMZ, provavelmente devido à inibição da proliferação celular. Em geral, os resultados do presente trabalho demonstraram que a estratégia de inibição do gene APE1 (participante da via BER) mostrou-se potencialmente viável, suportando a contribuição do BER na resistência à TMZ, visto que nas condições testadas, observou-se uma sensibilização das células de GBM, com efeito restrito às células de GBM resistentes (linhagem T98G), sendo pouco eficaz no sentido de sensibilizar as células sensíveis (linhagem U87MG) a esse agente. Assim, há que considerar as características genéticas de cada linhagem de GBM, visto que estas são cruciais para as respostas apresentadas pelas células aos tratamentos empregados. / APE1 (Apurinic/Apyrimidinic endonuclease 1/ Redox Factor-1 - APE1/REF-1) protein is a multifunctional enzyme whose expression is often increased in gliomas. Besides presenting activity in base excision repair (BER), APE1 also acts as a reduction factor, maintaining transcription factors (TFs) in an active reduced state. The BER pathway has been implicated as a possible factor of resistance to therapies based on the use of alkylating agents such as temozolomide (TMZ). In the present study, we have been using a strategy of small interference RNA (siRNA) to down-regulate the APE1 gene under conditions of treatment with TMZ in T98G (resistant to TMZ) and U87MG (sensitive to TMZ), glioblastoma (GBM), in order to determine the effects of APE1 gene silencing on cellular responses to this drug, evaluated by several assays, as well as the effects on the transcriptional expression of target genes of TFs regulated by APE1. APE1 silencing and TMZ treatment was effective to reduce the cell proliferation and clonogenic capacity of T98G cells, in addition to interfering in the cell cycle progression (S-phase arrest). These effects were accompanied by induction of DNA damage and phosphorylation of H2AX (H2AX), which may explain the decrease in cell survival. The same effect was not observed in silenced U87MG and TMZ-treated cells, being observed a predominance of the effects caused by TMZ itself, except for a slight induction of DNA damage and H2AX. Additionally, in silenced T98G and TMZ-treated cells, there was a moderate induction of apoptosis, as observed over time (1 to 10 days), with a slight induction of caspase-3 (on day 5); for those cells, we also observed autophagic induction (3.8 fold) at day 5. However, the induction of apoptosis and autophagy in U87MG cells was very low, suggesting that other mechanisms of cell death might be involved in the elimination of GBM cells under TMZ treatment. APE1 silencing caused a marked reduction on the invasiveness of T98G cells, similarly to that observed in TMZ treated cells, while the combination (APE1 silencing and drug treatment) led to an additive effect. For U87MG, the treatment combination was effective in reducing the proportion of invasive cells, in spite of an absence of any effect produced by each isolated condition tested. Regarding to the expression profile of target genes of TFs regulated by the APE1 redox activity, it was observed that COX2 and VEGF genes, targets of FTs NFB and HIF-1, were down-regulated in T98G while VEGF gene showed induced in U87MG cells; however, such alterations in the transcriptional expression pattern were observed only in response to TMZ treatment, independently of APE1 gene silencing, indicating no change in the APE1 redox activity, possibly due to the presence of APE1 remaining proteins inside cells. In addition, NFBp65(ser563) protein expression was increased in both cell lines (silenced and treated with TMZ), probably due to the reduced cell proliferation rates. In general, the present results show that the strategy of APE1 gene knockdown was potentially viable, supporting the BER contribution of the mechanism of TMZ resistance, since under the conditions tested, there was a sensitization of GBM cells. However, this effect was restricted to the resistant cell line (T98G cells). Thus, it should be considered the genetic characteristics of each GBM cell line, since these are crucial to the cellular responses to the conditions tested in the present work.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A emodina é uma antraquinona estruturalmente semelhante à aloe-emodina e ambas tem sido apontadas como capazes de causar lesões oxidativas pela produção de ERO. Sua presença em produtos dermocosméticos e de higiene pessoal, associada às informações de que a fotoativação de antraquinonas levaria ao aumento de lesões oxidativas causadas por ERO, torna relevante o estudo da associação da emodina com a radiação UVA. O objetivo desse trabalho foi avaliar a citotoxicidade induzida pela associação da emodina com doses subletais de radiação UVA, em células de Escherichia coli (selvagem e cepas deficientes em enzimas do BER), através de ensaios de sobrevivência bacteriana (taxa de dose de UVA igual a 20J/m/s, totalizando 108kJ/m ao final de 90min de experimento), e em células da linhagem A549 pela exclusão do corante azul de tripan e sobrevivência clonogênica(taxa de dose de UVA igual a 20J/m/s, totalizando 36kJ/m ao final de 30min de experimento). Além disso, a genotoxicidade desses agentes foi estudada por eletroforese em gel de agarose de DNA plasmidial (taxa de dose de UVA igual a 16J/m/s, totalizando 57,6kJ/m ao final de 60min de experimento). De acordo com os resultados: i) Concentrações iguais ou abaixo de 5,55mM de emodina não alteraram a sobrevivência em nenhuma das cepas estudas; ii) As proteínas Xth e Fpg parecem ter um papel importante no reparo das lesões causadas pela emodina, em altas concentrações, sugerindo a participação do reparo por excisão de bases (BER) nesse processo; iii) A associação da emodina com a radiação UVA se mostrou citotóxica em todas as cepas de E. coli; iv) O gene nfo foi o mais importante na resistência bacteriana às lesões induzidas pela associação dos dois agentes, reforçando o envolvimento do BER e indicando uma possível participação do reparo por incisão de nucleotídeos (NIR); v) A emodina parece ter interagido com o DNA plasmidial, alterando seu padrão de migração no gel de agarose; vi) Em células da linhagem A549, a emodina causa efeitos tóxicos imediatos que parecem ser reparados ao longo do tempo. Porém, quando a droga permaneceu por 24 horas em contato com as células, houve uma diminuição na sobrevivência celular que parece ser dosedependente; vii) As concentrações de 10μM e 25μM de emodina, quando associadas ao UVA, se mostraram responsáveis pela redução de mais de 50% na sobrevivência nas células A549, chegando a 100% de morte quando a concentração de emodina foi de 50μM; viii) A radiação UVA potencializou os efeitos citotóxicos da emodina, nos 2 modelos experimentais do presente estudo, indicando que a interação da emodina com a radiação UVA seja genotóxica e portanto prejudicial à saúde. / Emodin is an anthraquinone structurally similar to aloe-emodin and both have been identified as capable of causing oxidative damage by ROS production. Its presence in skin cosmetics and toiletries, associated to the information that the photoactivation of anthraquinones leads to increased oxidative damage caused by ROS, make studies about the association of emodin with UVA relevant. The aim of this study was to evaluate the cytotoxicity induced by the combination of emodin with sublethal doses of UVA radiation in Escherichia coli cells (wild strain and strains deficient in enzymes of BER) by bacterial survival assay (UVA dose rate equal to 20J/m/s, totaling 108kJ/m at the end of 90min of experiment); and in A549 cell line by trypan blue exclusion assay and clonogenic survival(dose rate of UVA equal to 20J/m/s, totaling 36kJ/m at the end of 30 minutes of experiment). Furthermore, the genotoxicity of these agents was studied by electrophoresis on agarose gel of plasmid DNA (dose rate of UVA equal to 16J/m/s, totaling 57,6kJ/m at the end of 60 minutes of experiment). According to the results: i) concentrations equal or below 5.55mM of emodin did not affect the survival in any of the studied strains; ii) proteins Xth and Fpg appear to have an important role in the repair of lesions induced by emodin, at high concentrations, suggesting the involvement of base excision repair (BER) in this process, iii) the association of emodin with UVA showed to be cytotoxic in all strains of E. coli iv) The nfo gene was the most important in bacterial resistance to damages induced by the association of the two agents, reinforcing the involvement of BER and indicating a possible role of the nucleotide incision repair (NIR), v) emodin appears to have interacted with plasmid DNA, altering its migration pattern in the agarose gel; vi) in A549 cell line, emodin caused immediate toxic effects that seemed to be repaired with time. However, when the drug remained for 24 hours in contact with the cells, there was a reduction in cell survival which seems to be in a dose dependent mode, vii) The concentrations of 10μM and 25μM of emodin, when associated with UVA, were responsible for a survival reduction of more than 50% survival in A549 cells, reaching 100% of death with the concentration of 50μM of emodin, viii) UVA radiation potentiates the cytotoxic effect of emodin in two experimental models in the present study, indicating that this interaction is genotoxic and therefore harmful to health.

DNA damage can cause genome instability, which may lead to human cancer. The most common form of DNA damage is DNA base damage, which is efficiently repaired by DNA base excision repair (BER). Thus BER is the major DNA repair pathway that maintains the stability of the genome. On the other hand, BER mediates DNA demethylation that can occur on the promoter region of important tumor suppressor genes such as Breast Cancer 1 (BRCA1) gene that is also involved in prevention and development of cancer. In this study, employing cell-based and in vitro biochemical approaches along with bisulfite DNA sequencing, we initially discovered that an oxidized nucleotide, 5’,2-cyclo-2-deoxyadenosine in DNA duplex can either cause misinsertion by DNA polymerase β (pol β) during pol β-mediated BER or inhibit lesion bypass of pol β resulting in DNA strand breaks. We then explored how a T/G mismatch resulting from active DNA demethylation can affect genome integrity during BER and found that pol β can extend the mismatched T to cause mutation. We found that AP endonuclease 1 (APE1) can use its 3'-5' exonuclease to remove the mismatched T before pol β can extend the nucleotide preventing a C to T mutation. The results demonstrate that the 3'-5' exonuclease activity of APE1 can serve as a proofreader for pol β to prevent mutation. We further explored the effects of exposure of environmental toxicants, bromate and chromate on the DNA methylation pattern on the promoter region of BRCA1 gene with bisulfite DNA sequencing. We found that bromate and chromate induced demethylation of 5-methylcytosines (5mC) at the CpG sites as well as created additional methylation at several unmethylated CpG sites at BRCA1 gene in human embryonic kidney (HEK) 293 cells. We further demonstrated that the demethylation was mediated by pol β nucleotide misinsertion and an interaction between pol β and DNA methyltransferase 1 (DNMT1) suggesting a cross-talk between BER and DNA methyltransferases. We suggest that DNA base damage and BER govern the interactions among the environment, the genome and epigenome, modulating the stability of the genome and epigenome and disease development.

Made available in DSpace on 2014-12-17T14:05:23Z (GMT). No. of bitstreams: 1 ThayseAS_TESE.pdf: 2133799 bytes, checksum: c236c1e72798e2c7e1008d1c78d046a4 (MD5) Previous issue date: 2013-03-21 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-?B and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-?B and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context / Estudos in vitro e em modelos animais sugerem que as prote?nas de reparo de DNA da via de reparo por excis?o de bases (do ingl?s, BER) APE1, OGG1 e PARP-1 est?o envolvidas tamb?m na resposta inflamat?ria. Neste trabalho foi investigado se os SNPs APE1 Asn148Glu, OGG1 Ser326Cys e PARP-1 Val762Ala associam-se ? meningite, e foi desenvolvido um sistema para an?lise funcional destas variantes polim?rficas. Os gen?tipos de pacientes com meningite bacteriana (MB), meningite ass?ptica (MA) e n?o infectados (controles) foram investigados por PIRA-PCR ou PCR-RFLP. Danos no DNA gen?mico foram detectados por meio de tratamento com Fpg. IgG e IgA foram titulados no plasma e citocinas e quimiocinas foram mensuradas em amostras de l?quor atrav?s de ensaios em Bio-Plex. Os n?veis de NF-?B e c-Jun foram dosados no l?quor dos pacientes por meio de dot blot. Foi observado um aumento significativo (P<0.05) na frequ?ncia do alelo APE1 148Glu nos casos de MB e MA. Os gen?tipos Asn/Asn no grupo controle e Asn/Glu no grupo da MB tamb?m apresentaram relevante aumento em suas frequ?ncias (P<0.05). Para o SNP OGG1 Ser326Cys, o gen?tipo Cys/Cys esteve mais frequente (P<0.05) nos casos de MB. A frequ?ncia do gen?tipo PARP-1 Val/Val foi mais alta no grupo controle (P<0.05). A ocorr?ncia combinada dos SNPs foi significativamente alta nos pacientes com MB, indicando que estes SNPs podem estar associados ? doen?a. Os portadores do alelo APE1 148Glu ou OGG1 326Cys apresentaram um n?mero maior de s?tios sens?veis ? Fpg, sugerindo que os SNPs afetam a atividade de reparo do DNA. Altera??es na s?ntese de IgG foram observadas na presen?a dos SNPs APE1 Asn148Glu, OGG1 Ser326Cys ou PARP-1 Val762Ala. Redu??es nos n?veis de IL-6, IL-1Ra, MCP-1/CCL2 e IL-8/CXCL8 foram encontradas na presen?a do alelo APE1 148Glu em amostras de pacientes com MB, no entanto n?o foram encontradas diferen?as nos n?veis de NF-?B e c-Jun considerando os gen?tipos e os grupos analisados. Utilizando APE1 como modelo, foi desenvolvido um sistema que possibilita a express?o e caracteriza??o funcional das enzimas polim?rficas estudadas e seus efeitos na c?lula, por meio de clonagem, utilizando o vetor pIRES2-EGFP e cDNA de APE1, transfec??o celular da constru??o obtida, inibi??o por siRNA de APE1 end?gena e genotipagem de culturas celulares. Em conclus?o, foram obtidas evid?ncias de um efeito significativo dos SNPs nos genes de reparo de DNA na regula??o da resposta imunol?gica. Este ? um trabalho pioneiro na ?rea, que demonstra a associa??o de variantes das enzimas da via BER com uma doen?a infecciosa em humanos, sugerindo que os SNPs estudados podem afetar a resposta imune e o impacto do n?vel de estresse oxidativo durante a infec??o cerebral. Desta forma, novos meios de an?lise funcional devem ser desenvolvidos para estudo de prote?nas polim?rficas e suas intera??es neste contexto / 2020-01-01

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O cloreto estanoso (SnCl2) e a radiação ultravioleta A (UVA) são agentes que lesam diversas estruturas celulares, inclusive o DNA, principalmente pela geração de espécies reativas de oxigênio. O objetivo deste trabalho foi estudar a mutagênese e o reparo das lesões produzidas pela combinação do UVA, na condição de préiluminação, com o SnCl2. Avaliou-se a ação de enzimas do reparo por excisão de bases (BER), em Escherichia coli (E. coli), por eletroforese em gel alcalino de agarose e sobrevivência bacteriana. Também se estudou a indução do sistema SoxRS pelo cromoteste, e a mutagênese pelo teste de Ames. De acordo com os resultados: i) o UVA induziu quebras no DNA das cepas testadas e os mutantes fpgnfo e fpg apresentaram maior retardo no reparo das lesões; ii) o SnCl2 induziu mais quebras que o UVA e os mutantes nfo e fpg mostraram maior dificuldade em reparar as lesões; iii) o UVA+SnCl2 provocou mais quebras que o SnCl2 e os mutantes nfo e fpg também apresentaram maior lentidão no reparo das lesões; iv) o UVA não inativou as cepas testadas; v) as cepas nfo e fpg foram as mais sensíveis ao SnCl2; vi) o UVA+SnCl2 provocou maior letalidade em todas as cepas testadas, em relação ao SnCl2, e os mutantes nfo e fpg também foram os mais sensíveis ao tratamento com ambos os agentes; vii) a transformação dos mutantes nfo com o plasmídio pBW21 (nfo+) e dos mutantes fpg com o plasmídio pFPG (fpg+) aumentou a sobrevivência das cepas aos tratamentos com SnCl2 e UVA+SnCl2; viii) o SnCl2 induziu o sistema SoxRS; ix) o SnCl2, UVA e UVA+SnCl2 não induziram mutagênese; x) o reparo das lesões parece ser preferencialmente realizado pelas proteínas Fpg e Nfo. / Stannous chloride (SnCl2) and ultraviolet radiation A (UVA) are able to induce lesions in different cellular structures, including DNA, manly through ROS generation. The aim of this work was to study the mutagenesis and repair of lesions induced by the association of UVA (pre treatment) with SnCl2. It was evaluated the action of base excision repair (BER) enzymes in Escherichia coli (E. coli) by alkaline gel electrophoresis and bacterial survival. It was also evaluated the SoxRS system induction by chromotest and mutagenesis through the Ames test. According to the results: i) UVA induced DNA strand breaks in all strains and fpg-nfo and fpg mutants showed greater delay in the repair of lesions; ii) SnCl2 induced more breaks than UVA and nfo and fpg mutants showed more difficult to repair the damage; iii) UVA + SnCl2 caused more breaks than the SnCl2 and nfo and fpg mutants also showed a slowest repair of injuries; iv) UVA did not inactivate any bacterial strains tested; v) nfo and fpg strains were more sensitive to SnCl2; vi) UVA + SnCl2 caused higher mortality in all strains tested, when compared to SnCl2, and, again, nfo and fpg mutants were the most sensitives to the treatment with both agents; vii) the transformation of nfo mutant with the plasmid pBW21 (nfo+) and fpg mutants with plasmid pFPG (fpg+) increased the survival of the strains to SnCl2 and UVA + SnCl2 treatments; viii) SnCl2 was able to induce SoxRS system; ix) SnCl2, UVA + SnCl2 and UVA did not induce mutagenesis; x) damage repair seems to be preferentially performed by Fpg and Nfo proteins.

FACT est une protéine clé, qui joue de multiples rôles, y compris dans la transcription et la réparation de l'ADN endommagé. Néanmoins, comment FACT participe à la réparation et à la transcription de la chromatine n'est pas élucidé. Dans ce travail nous avons tout d'abord étudié le rôle de FACT dans le processus de réparation par excision de base (BER). Nous avons utilisé des nucléosomes reconstitués avec de l'ADN à uracile incorporé au hasard. Nous avons trouvé que l'enzyme UDG est capable d'enlever les uraciles localisés du côté de la solution et pas les uraciles se trouvant en face de l'octamère d'histone. La présence simultanée de FACT et de RSC (facteur de remodelage de la chromatine, impliqué dans la réparation) permet un enlèvement efficace des uraciles localisés du côté de l'octamère d'histone par l'UDG. De plus, l'action concertée de FACT et RSC contribue à l'enlèvement de la lésion oxidative 8-oxoG, autrement inaccessible, de la matrice nucléosomale par l'enzyme OGG1. Ce résultat est obtenu grâce à une activité « co-remodelatrice » de la protéine FACT. Dans ce travail nous décrivons pour la première fois cette nouvelle propriété de FACT et nous montrons par une série d'expériences biochimiques que FACT est capable de stimuler l'activité de remodelage du RSC. Nos expériences montrent que la présence de FACT augmente l'efficacité de RSC à transformer l'énergie libérée par l'hydrolyse de l'ATP en travail « mécanique ». Les données obtenues suggèrent une nature stochastique du BER in vivo, FACT étant un facteur clé dans le processus de réparation. Nous avons également investigué l'implication de l'activité co-remodelatrice de FACT dans la fixation de NF-kB aux matrices nucléosomales. La production de nucléosomes remodelés, mais non - mobilisés (remosomes) n'est pas suffisante pour promouvoir la fixation de NF-kB. Pourtant, la mobilisation des nucléosomes par l'intermédiaire de RSC permet une interaction efficace entre NF-kB et l'ADN nucléosomal. Toutes ces données sont essentielles pour le décryptage du mécanisme moléculaire par lequel FACT agit dans le BER et dans la transcription médiée par NF-kB. / FACT is a vital protein which has multiple roles including one in transcription and repair of damaged DNA. However, how FACT assists repair and transcription remains elusive. In this work, we have first studied the role of FACT in Base Excision Repair (BER). We used nucleosomes containing DNA with randomly incorporated uracil. We found that the enzyme UDG is able to remove uracils facing the solution and not the uracils facing the histone octamer. The simultaneous presence of FACT and RSC (a chromatin remodeler involved in repair) allows, however, a very efficient removal of uracil facing the histone octamer by UDG. In addition, the concerted action of FACT and RSC permits the removal of the otherwise un-accessible oxidative lesion 8-oxoG from nucleosomal templates by OGG1. This was achieved thanks to the co-remodeling activity of FACT. Here we described for the first time this novel property of FACT and we show in a series of biochemical experiments that FACT is able to boost the remodeling activity of RSC. The experiments reveal that the presence of FACT increases the efficiency of RSC to transform the energy freed by ATP hydrolysis into “mechanical” work. The presented data suggest a stochastic nature of BER functioning in vivo, FACT being a key factor in the repair process. The implication of the co-remodeling activity of FACT in NF-kB factor binding to nucleosomal templates was also investigated. The generation of remodeled, but not mobilized nucleosomes (remosomes), was not sufficient to promote NF-kB binding. However, the RSC-induced nucleosome mobilization allows efficient NF-kB interaction with nucleosomal DNA. Our data are instrumental in deciphering the molecular mechanism of FACT implication in BER and NF-kB mediated transcription.

Made available in DSpace on 2015-03-03T15:19:19Z (GMT). No. of bitstreams: 1 AndreaLO.pdf: 448824 bytes, checksum: 4a360d0db40a607834f5c380870bc7f5 (MD5) Previous issue date: 2009-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The genome of all organisms is subject to injuries that can be caused by endogenous and environmental factors. If these lesions are not corrected, it can be fixed generating a mutation which can be lethal to the organisms. In order to prevent this, there are different DNA repair mechanisms. These mechanisms are well known in bacteria, yeast, human, but not in plants. Two plant models Oriza sativa and Arabidopsis thaliana had the genome sequenced and due to this some DNA repair genes have been characterized. The aim of this work is to characterized two sugarcane cDNAs that had homology to AP endonuclease: scARP1 and scARP3. In silico has been done with these two sequences and other from plants. It has been observed domain conservation on these sequences, but the cystein at 65 position that is a characteristic from the redox domain in APE1 protein was not so conservated in plants. Phylogenetic relationship showed two branches, one branch with dicots and monocots sequence and the other branch with only monocots sequences. Another approach in order to characterized these two cDNAs was to construct overexpression cassettes (sense and antisense orientation) using the 35S promoter. After that, these cassettes were transferred to the binary vector pPZP211. Furthermore, previously in the laboratory was obtained a plant from nicotiana tabacum containing the overexpression cassette in anti-sense orientation. It has been observed that this plant had a slow development and problems in setting seeds. After some manual crossing, some seeds were obtained (T2) and it was analyzed the T2 segregation. The third approach used in this work was to clone the promoter region from these two cDNAs by PCR walking. The sequences obtained were analyzed using the program PLANTCARE. It was observed in these sequences some motives that may be related to oxidative stress response / O genoma de todos os organismos est? sujeitos a les?es que podem ser causados por fatores end?genos e ambientais. Uma vez no genoma, as les?es podem levar a forma??o e ac?mulo de muta??es, as quais podem ser prejudiciais ao desenvolvimento do organismo. As vias de reparo de DNA s?o um mecanismo que permite o organismo detectar e corrigir essas les?es ou minimizar seus efeitos. V?rias vias de reparo de DNA s?o conhecidas e bem caracterizadas em animais e microorganismos. Em plantas, as vias de reparo ainda n?o s?o bem caracterizadas, mas muitas pesquisas t?m revelado a participa??o de todas as vias conhecidas no reparo do genoma das plantas, sendo os modelos mais estudados Arabidopsis thaliana e Oriza sativa. A via de reparo de interesse deste trabalho ? a via de BER, a qual apresenta v?rias prote?nas atuando no reparo do DNA. Por?m, a classe de prote?nas da via BER focadas s?o as AP endonucleases, respons?veis pela hidr?lise do s?tio AP. Este trabalho teve como objetivo caracterizar dois cDNAs de cana-de-a??car: scARP1 e scARP3, hom?logos a AP endonucleases de A.thaliana e identificados num trabalho de data-mining do projeto SUCEST. Para tanto, foram constru?dos cassetes de super-express?o, contendo o cDNA scARP1 sob o controle do promotor forte 35S. Al?m disso, anteriormente no laborat?rio foi obtido uma planta de icotiana tabacum contendo o cassete de super-express?o (35S+scARP1) em orienta??o anti-senso. Foi analisado o desenvolvimento desta planta, e foram obtidas tamb?m algumas sementes (T2) desta planta. Estas sementes foram germinadas e analisadas quanto ? presen?a do cassete de super-express?o e desenvolvimento. Al?m disso, para estes dois genes foram clonados as regi?es promotoras por PCR walking. Os fragmentos obtidos foram clonados, sequenciados e, analisados por meio do programa PLANTCARE. Os motivos encontrados nessas regi?es dos genes de cana-de-a??car foram comparados com potenciais regi?es promotoras das plantas de Arabidopis, O. sativa e S. bicolor. Estas an?lises mostraram a presen?a de diferentes motivos relacionados ? respostas ao estresse oxidativo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O cloreto estanoso (SnCl2) e a radiação ultravioleta A (UVA) são agentes que lesam diversas estruturas celulares, inclusive o DNA, principalmente pela geração de espécies reativas de oxigênio. O objetivo deste trabalho foi estudar a mutagênese e o reparo das lesões produzidas pela combinação do UVA, na condição de préiluminação, com o SnCl2. Avaliou-se a ação de enzimas do reparo por excisão de bases (BER), em Escherichia coli (E. coli), por eletroforese em gel alcalino de agarose e sobrevivência bacteriana. Também se estudou a indução do sistema SoxRS pelo cromoteste, e a mutagênese pelo teste de Ames. De acordo com os resultados: i) o UVA induziu quebras no DNA das cepas testadas e os mutantes fpgnfo e fpg apresentaram maior retardo no reparo das lesões; ii) o SnCl2 induziu mais quebras que o UVA e os mutantes nfo e fpg mostraram maior dificuldade em reparar as lesões; iii) o UVA+SnCl2 provocou mais quebras que o SnCl2 e os mutantes nfo e fpg também apresentaram maior lentidão no reparo das lesões; iv) o UVA não inativou as cepas testadas; v) as cepas nfo e fpg foram as mais sensíveis ao SnCl2; vi) o UVA+SnCl2 provocou maior letalidade em todas as cepas testadas, em relação ao SnCl2, e os mutantes nfo e fpg também foram os mais sensíveis ao tratamento com ambos os agentes; vii) a transformação dos mutantes nfo com o plasmídio pBW21 (nfo+) e dos mutantes fpg com o plasmídio pFPG (fpg+) aumentou a sobrevivência das cepas aos tratamentos com SnCl2 e UVA+SnCl2; viii) o SnCl2 induziu o sistema SoxRS; ix) o SnCl2, UVA e UVA+SnCl2 não induziram mutagênese; x) o reparo das lesões parece ser preferencialmente realizado pelas proteínas Fpg e Nfo. / Stannous chloride (SnCl2) and ultraviolet radiation A (UVA) are able to induce lesions in different cellular structures, including DNA, manly through ROS generation. The aim of this work was to study the mutagenesis and repair of lesions induced by the association of UVA (pre treatment) with SnCl2. It was evaluated the action of base excision repair (BER) enzymes in Escherichia coli (E. coli) by alkaline gel electrophoresis and bacterial survival. It was also evaluated the SoxRS system induction by chromotest and mutagenesis through the Ames test. According to the results: i) UVA induced DNA strand breaks in all strains and fpg-nfo and fpg mutants showed greater delay in the repair of lesions; ii) SnCl2 induced more breaks than UVA and nfo and fpg mutants showed more difficult to repair the damage; iii) UVA + SnCl2 caused more breaks than the SnCl2 and nfo and fpg mutants also showed a slowest repair of injuries; iv) UVA did not inactivate any bacterial strains tested; v) nfo and fpg strains were more sensitive to SnCl2; vi) UVA + SnCl2 caused higher mortality in all strains tested, when compared to SnCl2, and, again, nfo and fpg mutants were the most sensitives to the treatment with both agents; vii) the transformation of nfo mutant with the plasmid pBW21 (nfo+) and fpg mutants with plasmid pFPG (fpg+) increased the survival of the strains to SnCl2 and UVA + SnCl2 treatments; viii) SnCl2 was able to induce SoxRS system; ix) SnCl2, UVA + SnCl2 and UVA did not induce mutagenesis; x) damage repair seems to be preferentially performed by Fpg and Nfo proteins.