Characterization of the putative 9-1-1 complex of Leishmania major

Abstract

LmHus1 is a nuclear protein that is involved in DNA damage response in Leishmania major. The homologues of LmHus1 in other eukaryotes associate with the proteins Rad9 and Rad1 to compose the heterotrimeric complex 9-1-1. The 9-1-1 complex participate in the initial steps of DNA damage recognition and signaling. Our searches for putative partners of LmHus1 in L. major did not return any putative homologue of Rad1. On the other hand, we found a putative Rad9 homologue. We demonstrated that LmRad9 is a nuclear protein, which affect the parasite response to genotoxic drugs, suggesting its involvement in DNA damage response. It is noteworthy that LmRad9 structural characteristics suggest that LmRad9 can function as both Rad9 and Rad1 proteins. In fact, co-immunoprecipitation experiments demonstrated that LmRad9 interacts with LmHus1, which suggests the existence of a protein complex containing these two proteins. Therefore, the aim of our proposal is to characterize the putative 9-1-1 complex of L. major, formed by the proteins LmHus1 and LmRad9. In order to do that, we propose to investigate the native molecular mass of the parasite complex, the stequiometry of this interaction an the display of the two protein in the context of a protein complex. We believe that the information generated will contribute to a better understanding of the mechanisms possibly involved in the genome plasticity of these parasite.