Carolina Maria Berra
From DNA Repair Lab
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| == Resumo do Projeto == | == Resumo do Projeto == | ||
| - | O mecanismo de reparo por excisão de nucleotídeos (NER) é bastante conhecido por reparar lesões que causam distorção da dupla hélice de DNA, como as causadas por luz ultravioleta (UV). Contudo, algumas evidências vêm mostrando também a sua provável atuação em reparo de lesões oxidativas. Mutações em alguns genes envolvidos em NER estão associadas a doenças genéticas raras clinicamente distintas cujos fenótipos não podem ser explicados simplesmente por deficiência em reparo de lesões UV, mas podem estar associados à deficiência em reparo de lesões oxidativas. No entanto, o mecanismo exato da atuação de NER nesse tipo de lesão ainda permanece bastante controverso. Com o objetivo de uma melhor compreensão do envolvimento do NER em lesões oxidativas estamos averiguando os efeitos da fotoativação do azul de metileno (MB) em células deficientes e proficientes em NER. Sabe-se que a fotoativação do MB gera espécies reativas de oxigênio (ROS) que podem levar a formação de muitos tipos de danos oxidativos, como quebras simples, danos simples nas bases e adutos. Enquanto os danos oxidativos nas bases e quebras simples são normalmente formados pelo ataque direto de ROS no DNA, os adutos provavelmente resultam de produtos secundários formados a partir da reação de ROS com lipídeos e proteínas. Nossos dados mostram que células deficientes em NER (XPA e XPC), apesar de mostrarem a mesma cinética de reparo de bases oxidadas e de quebras formadas pela fotoativação do MB, apresentam grande sensibilidade ao estresse oxidativo gerado, além de serem muito mais susceptíveis à formação de quebras em seu DNA. Esses dados sugerem que a sensibilidade das células deficientes em NER se deve a problemas no reparo de adutos formados pelo estresse oxidativo e que as proteínas XPA e XPC parecem ter algum papel na proteção desse estresse, além da sua sabida participação em reparo de lesões induzidas por luz UV. | + | Nucleotide excision repair (NER) mechanism is well known to be involved in the removal of a variety of helix-distorting lesions, such as UV-induced lesions. However, the participation of this repair mechanism in oxidative DNA lesions is still controversial. Few data indicate that NER-deficient cells have problems to recognize and remove oxidized bases, suggesting the involvement of this pathway in oxidative lesion repair. In order to study the participation of NER mechanism in the removal of oxidative DNA lesions, we examined the effects of photoactivated methylene blue (MB) in NER-deficient and proficient cell lines. MB and light can produce reactive oxygen species (ROS). The reaction of ROS with DNA results in many kinds of oxidative damage, such as single base damage, bulky adducts and strand breaks. Whereas the small oxidative base damage and strand breaks are usually formed by a direct attack of ROS on DNA, the bulky adducts probably result from secondary products produced in reactions with lipids and proteins. Our results have shown that XP-A and XP-C NER-deficient cells are more sensitive to photoactivated MB-induced damage than wild type cells. In addition, using alkaline comet assay, XP-A and XP-C deficient cells were found to have more oxidative DNA lesions after MB and light treatment than wild type cells, although we have shown similarity in their repair kinetic. Therefore, these results suggest that XPA and XPC proteins may also have a role in the protection of cellular oxidative stress, in addition to its participation in repair of UV-induced DNA damage. |
| ==Publicações== | ==Publicações== | ||
Revision as of 20:17, 1 June 2007
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Bacharel e licenciada em ciências biológicas pela Universidade de São Paulo (USP). Mestre em microbiologia e biologia molecular (ICB-USP), hoje atua como aluna de doutorado pelo Depto. de Microbiologia-ICB-USP. |
Resumo do ProjetoNucleotide excision repair (NER) mechanism is well known to be involved in the removal of a variety of helix-distorting lesions, such as UV-induced lesions. However, the participation of this repair mechanism in oxidative DNA lesions is still controversial. Few data indicate that NER-deficient cells have problems to recognize and remove oxidized bases, suggesting the involvement of this pathway in oxidative lesion repair. In order to study the participation of NER mechanism in the removal of oxidative DNA lesions, we examined the effects of photoactivated methylene blue (MB) in NER-deficient and proficient cell lines. MB and light can produce reactive oxygen species (ROS). The reaction of ROS with DNA results in many kinds of oxidative damage, such as single base damage, bulky adducts and strand breaks. Whereas the small oxidative base damage and strand breaks are usually formed by a direct attack of ROS on DNA, the bulky adducts probably result from secondary products produced in reactions with lipids and proteins. Our results have shown that XP-A and XP-C NER-deficient cells are more sensitive to photoactivated MB-induced damage than wild type cells. In addition, using alkaline comet assay, XP-A and XP-C deficient cells were found to have more oxidative DNA lesions after MB and light treatment than wild type cells, although we have shown similarity in their repair kinetic. Therefore, these results suggest that XPA and XPC proteins may also have a role in the protection of cellular oxidative stress, in addition to its participation in repair of UV-induced DNA damage. PublicaçõesBerra C.M., Menck C.F.M. & Di Mascio P. Oxidative stress, genome lesions and signaling pathways in cell cycle control. Quím Nova, 29(6): 1340-1344, 2006. Morgante P.G *.; Berra C.M. *.; Nakabashi M.; Costa R.M.A.; Menck C.F.M. & Van-Sluys M.A. Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis. Gene, 344: 93-103, 2005. *These author contributed equaly to this work. Costa, R.M.A.; Morgante, P.G.; Berra, C.M.; Nakabashi, M.; Bruneau, D.; Bouchez, D.; Sweder, K.S.; Van Sluys, M.A. & Menck, C.F.M. The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development. Plant Journal, 28(4): 385-395, 2001. Costa, R.M.A.; Lima, W.C.; Vogel, C.I.G.; Berra, C.M.; Luche, D.D.; Medina-Silva, R.; Galhardo, R.S.; Menck, C.F.M. & Oliveira, V.R. DNA repair – related genes in sugarcane expressed sequence tags (ESTs). Genetics and Molecular Biology, 24 (1-4): 131-140, 2001. |
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