Characterization of LmRad1 of Leishmania major and study of its role in the DNA damage response

Abstract

The protozoan parasite Leishmania has a plastic and dynamic genome where gene amplification and chromosome translocation are relatively common phenomena. The molecular basis for these processes are likely to generate DNA structures that resemble damaged DNA. In higher eukaryotes, the cellular checkpoint control system detects these structures and blocks cell cycle progression, allowing for the required DNA repair. In mammalian and yeast cells, the trimeric complex 9-1-1 (Rad9, Rad1 and Hus1) participates in the initial steps of recognition and signaling of replicative stress. We have recently published the characterization of Leishmania major LmHus1 and LmRad9 proteins. We showed structural and phylogenetic similarities between these proteins and those from the 9-1-1 complex from other eukaryotes. The aim of this project is to investigate the existence of the missing Rad1 homologue in L. major. To do this, we propose to investigate the expression of a candidate homologue and study its role in the DNA damage response of the parasite. We will generate cell lines overexpressing LmRad1, as well as LmRad1 knockout parasites. The subcellular localization of the protein and its interaction with LmHus1 and LmRad9 will also be investigated. (AU)