Genes de recombinação envolvidos no reparo do DNA e na reprodução sexuada de Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the model organism studied in this work, has a high intraspecific genetic diversity which reflects on its phenotypic plasticity found in different hosts. The existence of naturally hybrid cells and the genetic recombination events observed in this parasite, besides the observation of specific meiosis genes in its genome, have raised questions about its exclusively clonal reproduction. In the present work we studied some genes involved in recombination processes, such as DMC1, RAD51 and ATM. After obtaining the modified T. cruzi parasites, we analyzed the phenotypic response of the cells when subjected to 500Gy of gamma radiation and to different genotoxic agents such as MMS and HU, both capable of generating DNA breaks. It was observed that cells deficient in ATM kinase activity were more sensitive to radiation and MMS than wild-type cells (CL Brener). Through expression of mRNAs of the genes analyzed, and by kinetic assays of phosphorylated histone H2A, the main marker of DSB in eukaryotes, we showed that there seems to be a co-regulation between ATM kinase and the specific T. cruzi DMC1 and RAD51 recombinases of homologous recombination. In assays to measure hybrid formation, using CellTrace dye, we showed, for the first time, that T. cruzi cells overexpressing the meiotic recombination gene (DMC1) became more apt for the cell fusion process. Based on structural biology analyses, we showed that the TcDMC1 protein seems have a high functional similarity to the human meiotic recombinase. This fact corroborates the high rate of hybrid formation observed in T. cruzi cells overexpressing DMC1. In view of the results observed throughout this work, we can suggest that DMC1, as in other organisms, is possibly also involved in meiotic recombination processes in Trypanosoma cruzi, which evidences a possible sexual reproduction in this parasite.