Estudo dos mecanismos moleculares da via de sinalização à resposta a danos ao DNA relacionada com o reparo acoplado à transcrição em Trypanosoma cruzi.

Abstract

Transcriptional stress which leads to RNA polymerase arrest can be caused by damage to the DNA. The stalled RNA polymerase is a significant signal to begin the transcription-coupled repair (TC-NER). Our previous results showed that high doses of UV kill the Trypanosoma cruzi rapidly and demonstrate that DNA lesions caused in the nuclear genome of the parasite can be persistent. In addition, studies of TcCSB, key protein in recognizing the stalled RNA polymerase, show that this protein is important for the parasite to deal with the DNA damage caused by genotoxic agents. Over-expression of TcCSB increased cell sensitivity to UV light in ATM/ATR kinase-dependent manner. On the other hand, TcCSB single-knockout cells showed resistance to UV light. These results suggest that the blockage of transcription is a stimulus to trigger a signaled death mechanism in T. cruzi. One of the objectives of this work was to verify if transcription blockage after UV damage is the principal cause of death, for this, the cells were pre-treated with α-amanitin and challenged with UV (1.500 J/m2). In addition, to investigate whether the sensitivity of T. cruzi to UV light is dependent on the cell cycle phase, the cells were synchronized with hydroxyurea (20mM) and the cell cycle determined by FACS, then, the cells were treated with UV (1.500 J/m²). Since, the DNA damage response pathway (DDR) during transcription results from the interaction with other proteins which are mediators. In this work, we investigated the role of TcRPA, TcRNAseH1, TcRNAseH2A mediators through the overexpression of these genes and we describe the phenotype of these strains after UV (1,500 J/m2). Finally, in this work, after obtaining the anti-TcCSB antibody, we performed the immunolocalization of the protein after irradiation with UV light (500 J/m2). In our results, we observe that cells pre-treated with a selective inhibitor of RNA polymerase II, which showed that these cells become resistant to UV light. However, when this compound was removed and the transcription process re-initiated, the cells went back to being sensitive to UV. Furthermore, our results allow us to conclude that G1 and G2 phases of the cell cycle affect the survival after exposure to UV light since in G1 phase an increase in death level occurs and phase G2 leads to a greater survival of the parasite. After UV irradiation, CSB protein was found concentrated in little foci located around the nucleus and in the kDNA for up to two hours. Differently, four hours after UV irradiation, the CSB protein presents a granular cytoplasmic distribution. Nevertheless, 24 hours after the irradiation, we did not observe any CSB labeling. Taken together, these data suggest that the death observed after exposure UV is dependent on RNA polymerase stalled in the initial phase of synthesis with the participation of TcRNApolII, TcCSB and TcATM and TcATR kinases according to the phase of the cell cycle. As well, it is proposed that the interaction of RPA-RNaseH1 and RPA-RNAseH2A is necessary to prevent causing DNA damage associated to R-loop and cell death. In conclusion, taken together, these data suggest that the TC-NER pathway is important for the parasite survival after UV in a way to ensure the maintenance of multigene transcription integrity.