Expression, purification and initial characterization of a new RPA-like protein in Trypanosoma brucei

Abstract

Parasitic protozoa of the Kinetoplastida order, which includes the genus Trypanosoma and Leishmania, are responsible for causing neglected diseases that results in a high number of deaths annually. Trypanosoma brucei, etiological agent of the sleeping sickness, is worldwide used as a model for studying protozoa biology due to the ease of cultivation and the wide range of tools and information available in literature. Despite the knowledge advances, big gaps in the processes that coordinate the DNA metabolism in these parasites still exist. Replication Protein A (RPA) is a key protein in the replication, transcription and DNA damage response machineries, in eukaryotes. This heterotrimetic protein, composed by subunits RPA-1, RPA-2 and RPA-3, is the major single-stranded DNA binding protein in eukaryotes. In trypanosomatids, this protein has already been described by our group and it is involved in replication and DNA damage response. Interestingly, the RPA-1 subunit of these protozoa doesn't possess the 70N domain, responsible for the interaction of RPA with a number of important proteins in the pathway of DNA repair and checkpoint signaling. This fact rise questions about the conservation of canonical pathways of DNA repair described in another eukaryotes. Through immunoprecipitation assays made by our group using RPA-2 as bait, a new RPA-like protein was found (containing a C-terminal domain similar to RPA-1) interacting with T. brucei RPA complex. This protein seems to be exclusive of trypanosomatids, and its role is still unknown. In this project, we intend to clone, express and purify this new T. brucei RPA-like protein as well as check its capability to bind single-stranded DNA and its participation in replication and DNA damage response in these organisms.