Caracterização funcional do gene Hus1 de Trypanosoma cruzi

Abstract

The DNA damage response (DDR) is a coordinated mechanism of damage signaling, in which lesion detection leads to cell cycle arrest and recruitment of repair machinery. One branch of the DDR is activated by the ATR kinase. In response to replication arrest, the 9-1-1 complex, formed by Rad9, Rad1 and Hus1, is loaded into single strand DNA regions covered by RPA. The presence of 9-1-1 facilitates the recruitment of TopBP1, allosteric activator of ATR. Active ATR is capable of phosphorylating the Chk1 kinase which in turn phosphorylates and inactivates Cdc25, promoting replication checkpoint and cell cycle arrest. Studies in Leishmania major suggest an association between Hus1 and increased capacity of this parasite in dealing with replicative stress. The mechanisms involved in the replicative stress response are not elucidated in Trypanosoma cruzi. Thus, this study aimed to characterize the Hus1 gene in T. cruzi, the parasite that causes Chagas disease. To study TcHus1 function was constructed a T. cruzi strain with increased levels of this gene. Thus, epimastigotes of CL Brener strain were transfected with the vector pROCKhigro containing the gene TcHus1. The analysis of overexpressor parasite growth profile under normal culture conditions suggested that overexpression of this gene does not compromise the basic functions of the cell. The growth profile of the parasites treated with replicative stress inducing drugs was analyzed. The treatment with benznidazole, cisplatin, camptothecin, hydroxyurea and methyl methane sulfonate (MMS) showed no difference in the growth profile between TcHus1 overexpressing cells and control cells. However, the quantitative real time PCR reaction of MMS treated cells showed that only pROCK-Hus1 parasites were able to increase Hus1 expression in response to replicative stress generated by MMS. The results presented in this study do not allow to conclude whether the function of Hus1 gene in T. cruzi is to promote the activation of the checkpoint kinase ATR, as describing in other eukaryotes. More studies are needed to understand the role and the importance of this pathway in the maintenance of genome integrity on this protozoan.