Investigação do reparo de dna acoplado ao processo de transcrição e recombinação na mitocôndria do Trypanosoma cruzi

• Ano de defesa: 2024
• Autor(a) principal: Wesley Roger Rodrigues Ferreira
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; Brasil; ICB - INSTITUTO DE CIÊNCIAS BIOLOGICAS; Programa de Pós-Graduação em Bioquímica e Imunologia; UFMG
• Palavras-chave em Português: Doença de chagas; Reparo de DNA; Mitocôndrias; T. cruzi; Bioquímica e Imunologia; Doença de Chagas; Trypanosoma cruzi; Reparo do DNA; Mitrocôndrias
• Link de acesso: http://hdl.handle.net/1843/78250; https://orcid.org/0000-0002-0891-630X

The Trypanosoma cruzi, the etiological agent of Chagas Disease, belongs to the Kinetoplastida order and possesses a unique and elongated mitochondrion. Although there is evidence of the presence of DNA repair pathways in the mitochondrial DNA (kDNA) of T. cruzi, the proteins involved in this process are not fully understood. In this study, we investigated the role of Rad51 proteins involved in the initiation of homologous recombination (HR) repair, Topo3α processing holliday junctions in HR, CSB protein involved in transcription-coupled nucleotide excision repair (TC-NER), and KAP7 protein, part of a unique family of trypanosomatid proteins associated with kDNA metabolism. In the search for evidence of mitochondrial HR, we used mt-Dox, a genotoxic agent inducing double-strand breaks in mitochondria. We observed sensitivity after 24 hours in TcRad51 (TcRad51+/-) heminocaut cells and Topo3α knockouts exposed to the same genotoxic agent. These cells ceased their growth, indicating that these proteins not only play a role in mitochondrial repair but also exhibit a differentiated response in DNA damage response (DDR) between the nucleus and mitochondria. Treating TcRad51+/- cells with cisplatin revealed sensitivity, and quantitative PCR of lesions showed more damage in kDNA. This indicates involvement in recombination repair in this organism. In the study of CSB protein function in the mitochondria, we found that CSB is involved in mitochondrial repair for lesions causing transcriptional arrest. We demonstrated that lesions generated by UV-C and cisplatin, typically repaired by TC-NER, are rapidly repaired in kDNA with CSB involvement. CSB overexpression accelerated repair, while depletion prolonged the presence of these lesions. Using the mitochondrial drug mt-Dox revealed distinct CSB signaling in the nucleus and mitochondria, with mitochondrial signaling reversible by nonspecific inhibition of DDR-involved kinases. Furthermore, we showed that CSB deficiency negatively impacts mitochondrial transcription after cisplatin exposure. Studying KAP7 protein, we collected evidence of its involvement in the mitochondrial DNA damage response. The results suggest that TcRad51 and TcTopo3α are involved in mitochondrial homologous recombination repair, indicating TcCSB involvement in mitochondrial transcriptional repair. These data point to the existence of a transcription-associated mitochondrial DNA repair pathway, differing from other organisms and possibly related to the presence of a single mitochondrion. Additionally, we observed differentiated mitochondrial signaling from the nucleus when the cell exclusively undergoes lesions in this organelle.