Chagas disease is caused by the protozoan Trypanosoma cruzi, which affect various regions of the world, with the main focus in Latin America. Despite advances, Chagas disease still causes about 40,000 new infections per year. The study of the molecular biology of this parasite may facilitate the discovery of new therapies and the development of antiparasitic drugs. Our group is studying the pre-replication and replication machineries of trypanosomes and, in this project, we intend to study the role of Orc1/Cdc6 and RPA, components of these machineries in the replication of T. cruzi chromosome ends. Chromosome ends comprise the telomeric complex which is formed by the interaction of DNA with proteins, and the latter can also interact with each other and are responsible for maintaining these termini. ORC complex is one of the components that form the pre-replication machinery along the chromosome, including telomeres and in T. cruzi, it is formed only by Orc1/Cdc6 protein. RPA is a trimeric complex divided into three subunits, which are found working independently or forming complexes with other proteins. They play several functions in DNA metabolism, being a key player in replication and telomere maintenance. In Leishmania spp., only the subunit 1 of RPA was found binding telomeric DNA and there is no description about the RPA-1 orthologue in trypanosomes. This study aims to understand the role played by both protein in the replication of T.cruzi chromosome termini. Specifically, we will clone, express and characterize the RPA-1 from this protozoa, and analyze its and Orc1/Cdc6's interactions with other components of the replication machinery at the C-telomeric strand using EMSA assays, the capture of proteins using biotinylated telomeric DNA affinity column, and co-localization of proteins and telomeres by FISH / IF. RNAi knockdown of T. brucei Orc1/Cdc6 will be use to check the importance of this interactions.
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