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Functional and Structural studies of LaRPA-1 protein in Leishmania amazonensis telomeres.

Grant number: 13/19089-1
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): December 01, 2013
Effective date (End): November 30, 2016
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal researcher:Maria Isabel Nogueira Cano
Grantee:Paula Maranon Garcia
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Telomeres are nucleoprotein complexes that protect the chromosomes ends in eukaryotes and they are maintained by telomerase. These structures provide stability and viability to the genome as they avoid chromosome terminal fusion and degradation. Telomere maintenance and telomerase activity depend on the action of proteins that associate with telomeric DNA. For example, in most eukaryotes the CST (CDC13-CTC1/Stn1/Ten1) complex interacts with telomerase and the DNA replication machinery, and controls the replication at the chromosome terminus. Leishmania spp. telomeres are composed by the conserved TTAGGG repeat, although a small number of telomeric proteins have been identified. The best-characterized telomeric complex is from L. amazonensis the main causative agent of Tegumentar leishmaniasis in the Americas, which is basically composed by three end-binding proteins: telomerase, LaRBP38 and LaRPA-1. LaRPA-1 binds and co-localizes with the G-rich telomeric strand in vivo and is probably the major parasite telomere end-binding protein. Recent results suggest that LaRAP-1 is probably a functional homologue of CDC13 and POT1, the end-binding proteins described respectively in yeast and vertebrates. These proteins belong to protein complexes, directly involved in the telomere-end protection (capping) and maintenance. In turn, these complexes control telomerase action and DNA replication at the chromosomes ends. This proposal aims to understand the role played by LaRPA-1 at parasite telomeres. We intend to generate parasites overexpressing full length LaRPA-1and the effects of these overexpression will be comparatively evaluated with wild parasites using different parameters as: proliferation of parasites in culture, protein expression and subcellular localization, qualitative and quantitative analysis of telomere length and chromatin imunoprecipitation (in vivo interaction with telomeres). We will test the role of LaRPA-1 during DNA replication by exposing wild type parasites and parasites over-expressing full length LaRPA-1 in the presence of DNA polymerase alpha inhibitors (e.g. aphidicolin). Recombinant proteins (wild type and truncated mutants) will be used to map putative protein-protein interaction domains using pull-down capture assays.