Functional dynamics of Orc1b protein during the life cycle of Trypanosoma

Abstract

Trypanosma cruzi is a protozoan which belongs to the Trypanosomatidae family and it is the etiological agent of Chagas Disease, a neglected disease that affects millions of people worldwide. This parasite alternates between replicative (epi and amastigotes) and non-replicative (trypomastigotes) forms in its hosts. Therefore it needs intense control of DNA replication, which involves coordinated action of several proteins. DNA replication starts with the assembly of the pre-replication complex (pre-RC) component ORC onto replication origins, located near the nucleosomes. Followed by the recruitment of other proteins from pre-RC, including Orc1b, whose function is not yet clear. Preliminary data of Marques, et al. (2016) indicates Orc1b participation as a positive regulator in the replication instead of having only a structural role in the ORC complex. Preliminary data from our group suggest the interaction of Orc1b with histones presenting specific post-translational modifications. Thus, we aim to deepen the studies of the Orc1b role in the DNA replication of T. cruzi and its possible interactors. Assays will be performed to analyze the expression of this protein in the course of T. cruzi cell and life cycle, and also the production of hemi knockouts to access the impact of the absence of this protein in the replication. In addition, immunoprecipitation, pull down, ChIP-seq and ELISA assays will be executed in order to analyze the possible interactors of Orc1b, with emphasis on the modified histones H4K10ac and H3K76Me3. This knowledge will support the development of future antiparasitic strategies and it will also be useful to further understand the biology of these parasites. (AU)