Identification of LmRad9, LmHus1 and LmRad1-interacting proteins and generation of a LmHus1 conditional knockout cell line

Abstract

Eukaryote cells have developed mechanisms to coordinate DNA damage sensing, cell cycle arrest and DNA damage repair in order to grant genome stability. In higher eukaryotes, the 9-1-1 checkpoint complex, composed by the proteins Rad9, Rad1 and Hus1, participates in the early steps of the DNA damage response. We have identified possible homologues of each one of the three subunits of the 9-1-1 complex in Leishmania major. Our experiments demonstrate that LmHus1 and LmRad9 are involved in telomere maintenance, genotoxic stress response and cell cycle progression control, indicating their involvement in genome stability maintenance mechanisms in this parasite. We have also demonstrated that LmHus1 and LmRad9 are present in protein complexes and interact with each other in vivo. It suggests that 9-1-1 complex might had been conserved in this parasite. However, we have also observed putative sequence and structural divergence of the 9-1-1 complex subunits in L. major, as compared to eukaryotic counterparts, and we wish to understand the significance of these observations. Leishmania displays remarkable genome plasticity that may be exploited not only for control of gene expression, but also likely in infection progression. The structural divergence of the 9-1-1 complex could underlie the mechanisms that mediated the genome plasticity observed in this parasite. Therefore, elucidating the structure and function of 9-1-1 complex it is a mandatory step to understand the evolution of this machinery in eukaryotic cells. Besides, it could help us to understand the strategies used by this parasite to establish infection. (AU)