Project title, identification of possible interactions among the DNA repair machinery and telomere maintenance in Leishmania spp.

Abstract

Telomeres are DNA. protein complexes that protect the end of the chromosomes. They confer genome stability and cell viability by avoiding terminal fusion and chromosome degradation. Leishmania spp. telomeres are composed by TTAGGG repeats which are maintenad by telomerase. Proteins that associate with telomeric DNA are crucial components that help to maintain telomere architeture and to regulate telomerase activity. Among these proteins are RPA-1 and TRF, which will be in part focus of the present study. These proteins play multiple roles in eukaryotic DNA metabolism, including telomere maintenance and DNA repair. In our lab, LaRPA-1 was identified and characterized as a protein that has high affinity for the telomeric G rich single-strand and that co-localizes in vivo with parasite telomeres. Recent results show that in some eukaryotes RPA function as a DNA damage sensor at telomeres. It interacts with telomeres during damage and recruits the recombinase RAD51 to initiate a local repair. LaTRF also interacts in vitro and in vivo and co-localizes with parasite telomeres. The vertebrate TRF homologues interact with some DNA repair machinery components in a cell cycle-dependent manner. The present work has the aim to identify the possible interactions of some Leishmania telomeric proteins (i.e. LaRPA-1, LaTRF and telomerase) with components of the DNA repair machinery. To achieve this goal parasites will be submitted to treatment with different genotoxic agents (i.e. phleomycin and hydrogen peroxide). Alterations in telomere size will be estimated by non-denaturing Southern blot and variations in gene and protein expression will be verified using RT-PCR and Western blotting. Imunoprecipitation and imunofluorescence coupled with FISH will be respectively used to check possible protein:protein interactions and proteins subcellular co-localization. These results may be confirmed by pull-down and co-imunoprecipitation assays, followed by mass espectrometry analysis. (AU)