Navegando por Palavras-chave "DNA repair"

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    Biochemical studies with DNA polymerase beta and DNA polymerase beta-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance
    (Elsevier B.V., 2008-11-01) Lopes, Debora de Oliveira; Fonseca Schamber-Reis, Bruno Luiz; Regis-da-Silva, Carlos Gustavo; Rajao, Matheus Andrade; DaRocha, Wanderson Duarte; Macedo, Andrea Mara; Franco, Gloria Regina; Nardelli, Sheila Cristina [UNIFESP]; Schenkman, Sergio [UNIFESP]; Hoffmann, Jean-Sebastein; Cazaux, Christophe; Junho Pena, Sergio Danilo; Ribeiro Teixeira, Santuza Maria; Machado, Carlos Renato; Universidade Federal de Minas Gerais (UFMG); Universidade Federal de São Paulo (UNIFESP); CNRS
    Mammalian DNA polymerase p is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. in trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerase beta, TcPol beta and TcPol beta PAK, and showed that both enzymes localize to the parasite kinetoplast. in vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPol beta PAK, in comparison to TcPol beta, conducted DNA synthesis over a much broader pH range. TcPol beta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPol beta were not observed for TcPol beta PAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPol beta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis. (C) 2008 Elsevier B.V. All rights reserved.
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    Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions
    (Elsevier B.V., 2012-01-01) Moraes, Maria Carolina S.; Andrade, Annabel Quinet de; Carvalho, Helotonio [UNIFESP]; Guecheva, Temenouga; Agnoletto, Mateus H.; Henriques, Joao A. P.; Sarasin, Alain; Stary, Anne; Saffi, Jenifer; Menck, Carlos F. M.; Universidade de São Paulo (USP); Univ Paris Sud; Universidade Federal de São Paulo (UNIFESP); Univ Fed Rio Grande do Sul; Fed Univ Hlth Sci Porto Alegre UFCSPA
    Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. in fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
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    Chromatin and nuclear organization in Trypanosoma cruzi
    (Future Medicine Ltd, 2009-10-01) Elias, Maria Carolina; Nardelli, Sheila Cristina [UNIFESP]; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst Butantan
    A total of 100 years have passed since the discovery of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas' disease. Since its discovery, the molecular and cellular biology of this early divergent eukaryote, as well as its interactions with the mammalian and insect hosts, has progressed substantially. It is now clear that this parasite presents unique mechanisms controlling gene expression, DNA replication, cell cycle and differentiation, generating several morphological forms that are adopted to survive in different hosts, in recent years, the relationship between the chromatin structure and nuclear organization with the unusual transcription, splicing, DNA replication and DNA repair mechanisms have been investigated in T. cruzi. This article reviews the relevant aspects of these mechanisms in relation to chromatin and nuclear organization.
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    Correlation of MLH1 and MGMT expression and promoter methylation with genomic instability in patients with thyroid carcinoma
    (Biomed Central Ltd, 2013-02-15) Santos, Juliana Carvalho; Bastos, Andre Uchimura [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; Ribeiro, Marcelo Lima; Univ Sao Francisco; Universidade Federal de São Paulo (UNIFESP)
    Background: Gene silencing of the repair genes MLH1 and MGMT was shown to be a mechanism underlying the development of microsatellite instability (MSI), a phenotype frequently associated with various human malignancies. Recently, aberrant methylation of MLH1, MGMT and MSI were shown to be associated with mutations in genes such as BRAF, RAS and IDH1 in colon and brain tumours. Little is known about the methylation status of MLH1 and MGMT in thyroid tumours and its association with MSI and mutational status.Methods: in a series of 96 thyroid tumours whose mutational profiles of BRAF, IDH1 and NRAS mutations and RET/PTC were previously determined, we investigated MLH1 and MGMT expression and methylation status by qPCR and methylation-specific PCR after bisulphite treatment, respectively. MSI was determined by PCR using seven standard microsatellite markers.Results: Samples with point mutations (BRAF, IDH1 and NRAS) show a decrease in MLH1 expression when compared to negative samples. Additionally, malignant lesions show a higher MSI pattern than benign lesions. the MSI phenotype was also associated with down-regulation of MLH1.Conclusions: the results of this study allow us to conclude that low expression of MLH1 is associated with BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS) in patients with thyroid carcinoma. in addition, a significant relationship between MSI status and histological subtypes was found.
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    DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells
    (Springer, 2010-04-01) Carvalho, Helotonio [UNIFESP]; Garrido, Leandro M.; Furlan, Renata L. A.; Padilla, Gabriel; Agnoletto, Mateus; Guecheva, Temenouga; Henriques, Joao A. P.; Saffi, Jenifer; Martins Menck, Carlos Frederico; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Univ Fed Rio Grande do Sul
    Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. the anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene.We assessed the induction of apoptosis (Sub-G(1)) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay).Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. the effects of cosmomycin D were equivalent to those obtained with doxorubicin. the broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay.Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.
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    DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions
    (Elsevier B.V., 2012-06-01) Schamber-Reis, Bruno Luiz Fonseca; Nardelli, Sheila Cristina [UNIFESP]; Regis-Silva, Carlos Gustavo; Campos, Priscila Carneiro; Cerqueira, Paula Gonçalves; Lima, Sabrina Almeida; Franco, Gloria Regina; Macedo, Andréa Mara; Pena, Sergio Danilo Junho; Cazaux, Christophe; Hoffmann, Jean-Sebastien; Motta, Maria Cristina Machado; Schenkman, Sergio [UNIFESP]; Teixeira, Santuza Maria Ribeiro; Machado, Carlos Renato; Universidade Federal de Minas Gerais (UFMG); Universidade Federal de São Paulo (UNIFESP); Universidade Federal do Rio de Janeiro (UFRJ); Super Learning & Dev Ctr CESED; CNRS; Univ Toulouse
    Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. in mammals, DNA polymerase beta (Pol beta) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpol beta) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpol beta showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpol beta overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpol beta was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpol beta during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle. (C) 2012 Elsevier B.V. All rights reserved.
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    Modificações em marcas epigenéticas em histonas ao longo da gênese do melanoma
    (Universidade Federal de São Paulo (UNIFESP), 2011) Meliso, Fabiana Marcelino [UNIFESP]; Jasiulionis, Miriam Galvonas [UNIFESP]
    Entre os principais transtornos mundiais, o cancer permanece como a segunda doenca responsavel pelo maior numero de mortes no mundo e o melanoma e, cada vez mais, um dos tipos mais preocupantes de tumor. Os tumores, como amplamente estudado, estao relacionados a fatores geneticos e epigeneticos, entretanto, ate o momento, nao esta claro quais sao os fatores que levam ao estabelecimento de padroes epigeneticos aberrantes associados tanto ao inicio quanto a progressao tumoral. Recentes estudos tem demonstrado que alteracoes no microambiente celular, assim como o estresse oxidativo, sao importantes fatores que levam ao estabelecimento de caracteristicas neoplasicas. Metilacao de DNA e modificacoes pos-traducionais em histonas sao os mecanismos epigeneticos mais estudados. Embora haja estudos sobre metilacao de DNA aberrante em melanomas, ainda sao escassos os estudos sobre marcas em histonas nestes tumores. Nesse sentido, utilizando um modelo murino de transformacao maligna de melanocitos associado a condicoes sustentadas de estresse, esse trabalho teve por objetivo identificar padroes epigeneticos aberrantes associados a diferentes fases da genese do melanoma. Foram observadas variacoes em marcas de histonas relacionadas a ativacao ou repressao genica nas primeiras etapas e ao longo da progressao maligna, com indicios de cromatina aberta nos estagios intermediarios da transformacao. Da mesma forma, identificamos alteracoes em componentes da maquinaria epigenetica ao longo da transformacao maligna dos melanocitos. Alem disso, dados ainda preliminares indicam associacao diferencial de sequencias de DNA a desacetilase de histonas Sirt1 apos submeter os melanocitos a situacao de estresse sustentado, o que pode indicar um papel de Sirt1 no estabelecimento de padroes epigeneticos aberrantes. Finalmente, esse trabalho mostra mudancas dinamicas tanto em componentes da maquinaria quanto em marcas epigeneticas ao longo da genese do melanoma e ainda sugere reprogramacao epigenetica e aquisicao de caracteristicas de pluripotencia nos estagios que precedem a aquisicao do fenotipo maligno pelos melanocitos
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    Subtelomeric I-Scel-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi
    (Frontiers Media Sa, 2016) Chiurillo, Miguel A.; Moraes Barros, Roberto R.; Souza, Renata Torres [UNIFESP]; Marini, Marjorie Mendes [UNIFESP]; Antonio, Cristiane Regina [UNIFESP]; Cortez, Danielle Rodrigues [UNIFESP]; Curto, Maria A.; Lorenz, Hernan A.; Schijman, Alejandro G.; Ramirez, Jose L.; Silveira, Jose Franco da [UNIFESP]
    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a I cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-Scel meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.
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    Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis
    (Elsevier B.V., 2008-06-01) Carvalho, Helotonio [UNIFESP]; Ortolan, Tatiana G.; dePaula, Tomas; Leite, Ricardo A.; Weinlich, Ricardo; Amarante-Mendes, Gustavo P.; Martins Menck, Carlos Frederico; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    p53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATWATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. the levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling. (c) 2008 Elsevier B.V. All rights reserved.