Trypanosoma cruzi is the etiologic agent of Chagas' disease. Despite being the largest cause of heart disease-induced infection, there is no vaccine, and the only available drugs have serious side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology. During the life cycle, T. cruzi alternates between proliferative (amastigote and epimastigote), and non-proliferative forms (trypomastigote and metacyclic trypomastigote). In addition, the development of dormant amastigote forms that are non-proliferating and resistant to treatment has recently been demonstrated. Our laboratory studies DNA replication in trypanosomes, and has demonstrated that the Orc1/Cdc6 protein is a component of the pre-replication complex (PRC). In eukaryotes, this complex recognizes and associates with origins of replication, nucleating the process of genomic duplication at specific points of DNA. In trypanosomes, our group showed that Orc1/Cdc6 remains associated with DNA throughout the cell cycle of the epimastigote form and also interacts with DNA in the amastigote form. However, in the trypomastigote and metacyclic trypomastigote forms, unable to duplicate the genetic material, Orc1/Cdc6 is located in nuclear space, but is unable to interact or weakly interact with DNA, a fact that should contribute for lacking of replication in these forms. This project aims to evaluate the interaction of Orc1/Cdc6 with DNA in dormant amastigote forms in order to generate an overview of the Orc1/Cdc6-DNA interaction throughout the life cycle of T. cruzi. Next, we intend to look for possible mechanisms that may inhibit the Orc1/Cdc6-DNA interaction in non-replicative forms. Our approach will be to search for Orc1/Cdc6 posttranslational modifications (PTM) exclusively in the replicative or non-replicative forms of T. cruzi and to verify the possible role of these PTMs in the Orc1/Cdc6-DNA interaction. In addition, we intend to search for Orc1/Cdc6 interactors during the life cycle of this parasite in order to identify possible modulators of the Orc1/Cdc6-DNA interaction. Therefore, we intend to purify Orc1/Cdc6 from the different forms of T. cruzi and to evaluate by mass spectrometry (i) the PTM and (ii) the Orc1/Cdc6 interactors. The data obtained will be validated through generations of specific lineages and further Orc1/Cdc6-DNA interaction analysis.
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