Síntese translesão e reparo de DNA em Trypanosoma cruzi: caracterização funcional da DNA polimerase kappa e análise da remoção de lesões no DNA nuclear e mitocondrial

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease, and during its life cycle it is subjective to several genotoxic agents, which can lead to mutations, cell division blockage or even cell death. The major mechanisms of DNA damage tolerance are the DNA repair (which removes the lesion) and the translesion synthesis (TLS, carried out by specialized DNA polymerases that are able to replicate across DNA damage). Within this thesis, we characterized one copy of the T. cruzi DNA polymerase kappa gene (TcPOLK-2), as well as we assessed the T. cruzi DNA repair after treatment with different genotoxic agents. Our results showed that TcPol-2 is a mitochondrial DNA polymerase which can replicate across 8-oxoguanine lesions and also can synthesize DNA in a recombination intermediate structure. These TcPol-2 specializations that were verified in vitro reflected on in vivo phenotypes, since the overexpression of TcPol-2 increased the T. cruzi resistance against agents that cause oxidative damage and double-strand breaks. These results suggest a TcPol-2 role in the translesion synthesis across oxidative damage and in the mitochondrial DNA recombination. Additionally, we demonstrated that the other TcPol copy (TcPol-1) is localized to the nucleus. When we assessed the DNA repair of T. cruzi after different genotoxic treatments, we verified that T. cruzi has nuclear and mitochondrial mechanisms of DNA repair. Our results showed that T. cruzi efficiently repairs DNA lesions caused by MMS and cisplatin, removing both nuclear and mitochondrial lesions. We also verified that H2O2- induced lesions are efficiently removed from nuclear DNA, whereas the mitochondrial lesions persist. The persistence of mitochondrial lesions showed to be a consequence of a defect in the mitochondria activity, which leads to a second burst of oxidative species. We also verified that T. cruzi is capable of repairing UV-induced lesions in the mitochondrial DNA, while nuclear lesions remained unrepaired. This surprising mitochondrial repair of UV and cisplatin-induced lesions seems to be carried out by a mechanism similar to the interstrand crosslink repair, since proteins belonging to this pathway are localized to the kinetoflagellar zone adjacent to the mitochondrial DNA. Taken together, the results obtained herein show that the maintenance of mitochondrial DNA is critical for T. cruzi survival, and this can be achieved even by translesion synthesis mechanisms as by different mitochondrial DNA repair pathways.