O sistema GO em Trypanosoma cruzi: caracterização dos genes TcOGG1 e TcMYH

• Ano de defesa: 2013
• Autor(a) principal: Marianna Kunrath Lima
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Bioquímica e Imunologia; Tripanossoma cruzi; Bioquímica; Estresse oxidativo
• Link de acesso: http://hdl.handle.net/1843/BUBD-9P3JHB

Trypanosoma cruzi is a protozoan parasite, causative of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and needs to adapt to distinct environments. Hence, DNA repair is essential for its survival and improvement of infection (Aguiar et al., 2013). Given the importance of DNA repair to T. cruzi, we decided to study if this protozoan has homologous of the enzymes OGG1 (TcOGG1) and MYH (TcMYH). First, we performed heterologous complementation assays. For OGG1, experiments were carried out in Escherichia coli, however, expression of TcOGG1 was toxic both to wild type (WT) cell (AB1157) and to fpg -/- cells (BH20). Nevertheless, expression of TcOGG1 was not toxic to Saccharomyces cerevisiae. Yeast mutants ogg1-/- (CD138) have an increased spontaneous mutation frequency, but when they expressed TcOGG1, this frequency was similar to the one seen for the WT cell (FF18733). On the other hand, TcMYH was not toxic to E. coli and complemented the bacterial MutY- strain, reducing the mutation frequencies to a level similar to the wild types frequencies. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed T. cruzi lineages that overexpress OGG1 or MYH. Although in standard conditions these lineages have similar growth in comparison with control cells, the overexpressors are more sensitive to hydrogen peroxide than control. When overexpressor parasites were treated with benzonidazole, the drug used in Chagas disease treatment, they exhibited no phenotype. Also, TcOGG1-overexpressor T. cruzi exhibited lower levels of DNA damage in the nucleus, but not in the kDNA (kinetoplast (mitochondrial) DNA). This might be related to the quantity of the enzyme present in each subcellular compartment and to the unequal distribution of other repair proteins in the nucleus and kDNA. Localization experiments with TcOGG1 or TcMYH fused to GFP showed that the protozoans enzymes are both nuclear and mitochondrial.These data suggest that T. cruzi has functional 8-oxoguanine and MutY DNA glycosylases, which participate in nuclear and mitochondrial BER.