|
LaTBP1: A Leishmania amazonensis DNA-BINDING PROTEIN THAT ASSOCIATES IN VIVO WITH TELOMERES AND GT-RICH DNAs USING A MYB-LIKE DOMAIN
Cristina B.B. Lira1,2, Jair L. Siqueira Neto1,2, Letícia Khater2,3, Thiago C. Cagliari 2,3, Luis A. Peroni2, José R.R. dos Reis2, Carlos H.I. Ramos3 and Maria I.N. Cano1
1Departamento de Genética, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brazil; 2 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil; 3Laboratório Nacional de Luz Síncrotron Campinas, SP, Brazil .
Telomeric DNA-binding proteins (TBP) are crucial cellular components that regulate the structure and function of eukaryotic telomeres. An important TBP is the multifunctional scRap1p, which binds the yeast TG1-3 double-stranded telomeric DNA and was first described as a transcription factor. scRap1 orthologs are found in unicellular and multicellular eukaryotes with low sequence similarities. Rap1 proteins are characterized by the presence of three functional domains: BRCT, RCT, and the Myb-like DNA-binding motif. A L. major protein, containing most of the Rap1 functional domains, was identified by data-mining search and a homologous gene was cloned from L. amazonensis (LaTBP1). The recombinant LaTBP1, bound the double-stranded Leishmania telomeric DNA and other GT-rich sequences in vitro. LaTBP1 was transcribed and translated in Leishmania as shown by RT-PCR and Western Blot assays. DNAse I footprinting defined the boundaries of LaTBP1 telomeric DNA-binding site in vitro and chromatin immunoprecipitation showed that LaTBP1 bound in vivo both telomeric and GT-rich sequences. It was also possible to show that the putative LaTBP1 Myb domain possesses a hydrophobic cavity nearby the solely Trp residue, as shown by emission fluorescence spectroscopy and by comparative modeling. This cavity was shown to be implicated in DNA-binding activity. The possibility of LaTBP1 being a Leishmania Rap1 orthologue is discussed.
Financial Support: FAPESP, CNPq, UNICEF/ UNDP/World Bank/WHO (TDR).
|