Identification and characterization of novel components of the telomeric complex of tripanosomatids using in vitro and in vivo studies.

• Grant number: 11/19682-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: March 01, 2012
• End date: February 28, 2014
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Maria Isabel Nogueira Cano
• Grantee: Vinícius Santana Nunes
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Telomeres are protein:DNA complexes that protect the end of the chromosomes. Telomeres maintenance and telomerase activity depend on protein complexes that associate with telomeric DNA. For example, the CST complex interacts with the G-rich telomeric strand and with the DNA replication machinery to regulate telomerase activity and control DNA replication at the chromosome ends. Trypanosomatid telomeres are composed by (TTAGGG)n repeats however, few telomeric proteins specific to these organisms have been identified so far. The best characterized trypanosomatid telomeric complex is that of L. amazonensis, which consists basically of telomerase, LaRBP38 and LaRPA-1 that bind the 3´G-overhang and probably are functional homologues of the budding yeast and mammalian CST components. The hypothesis that we will test, and which guides this proposal, is based on the fact that LaRPA-1 is probably involved in telomere length maintenance and capping in L. amazonensis and perhaps in others trypanosomatids. In most eukaryotes, RPA is a heterotrimeric complex of single-stranded DNA-binding proteins that play multiple roles in DNA metabolism, including telomere maintenance and DNA repair. In Leishmania, LaRPA-1 was found binding the G-rich telomeric strand. Moreover, the biology of telomeres in these parasites may involve telomeric proteins still not characterized. Using in silico analysis, we propose to identify homologues of the TEN1 e STN1 that are part of the CST complex in other organisms, which will be characterized fused with GFP. In collaboration with Dr. Tzafati from Israel, we will incorporate the trypanosomatid telomeric sequence onto K. lactis telomeres, and characterize the effects of this incorporation on telomere maintenance and capping. Finally, we will try to identify and characterize in vitro proteins that interact with LaRPA-1/TcRPA-1. The efficiency of the yeast heterologous system and the biochemical assays, will be valuable approaches to understand the conservation of telomeres biology in trypanosomatids.