Functional characterization of Leishmania Rad1, a subunit of the 9-1-1 checkpoint complex

Abstract

Leishmaniasis is a parasitic disease of undeniable medical importance. The etiological agent, Leishmania, possesses mechanisms of adaptation frequently associated with genomic plasticity, including variation in the number of chromosome and gene copies. These adaptions may arise as a consequence of DNA replication stress and activation of DNA damage repair pathways. Our laboratory studies Leishmania factors involved in the replicative stress response, including the 9-1-1 complex (Rad9-Hus1-Rad1), that acts during the initial steps of the response and regulates cell cycle progression. The Rad9 and Hus1 subunits have been studied in more detail however, the characterization of the third subunit Rad1 remains limited. In mammals, Rad1 depletion coincides with chromosomal abnormalities, suggesting both an independent role for Rad1 in meiotic cells, and a broader range of functions for this protein in genome stability. This proposal aims to characterize the function of Rad1 in Leishmania using genome-edited cell lines using CRISPR/Cas9, to explore Rad1's subcellular localization and analyze the effects of its deletion. To do this, we will study Rad1 in the context of the replicative stress response signaling, examining for the accumulation of single-strand and double-strand DNA breaks, amplification of extrachromosomal elements, and copy number variations in Rad1-deficient or null cell lines. Understanding Rad1's role in these parasites is crucial for elucidating important aspects of Leishmania biology and may pave the way for the development of new strategies to control this neglected pathogen.