Leishmania braziliensis replication protein A subunit 1: Molecular modelling,protein expression and analysis of its affinity for both DNA and RNA

Background: replication factor a (rpa) is a single-strand dna binding protein involved in dna replication,recombination and repair processes. it is composed by the subunits rpa-1,rpa-2 and rpa-3; the major dna-binding activity resides in the subunit 1 of the heterotrimeric rpa complex. in yeast and higher eukaryotes,besides the three basic structural dna-binding domains,the rpa-1 subunit contains an n-terminal region involved in protein-protein interactions with a fourth dna-binding domain. remarkably,the n-terminal extension is absent in the rpa-1 of the pathogenic protozoan leishmania (leishmania) amazonensis; however,the protein maintains its ability to bind ssdna. in a recent work,we identify leishmania (viannia) braziliensis rpa-1 by its specific binding to the untranslated regions of the hsp70 mrnas,suggesting that this protein might be also an rna-binding protein. methods: both rlbrpa-1 purified by his-tag affinity chromatography as well as the in vitro transcribed l. braziliensis 3′ hsp70-ii utr were used to perform pull down assays to asses nucleic acid binding properties. also,homology modeling was carried out to construct the lbrpa-1 tridimensional structure to search relevant amino acid residues to bind nucleic acids. results: in this work,after obtaining the recombinant l. braziliensis rpa-1 protein under native conditions,competitive and non-competitive pull-down assays confirmed the single-stranded dna binding activity of this protein and demonstrated its interaction with the 3′ utr from the hsp70-ii mrna. as expected,this protein exhibits a high affinity for ssdna,but we have found that rpa-1 interacts also with rna. additionally,we carried out a structural analysis of l. braziliensis rpa-1 protein using the x-ray diffraction structure of ustilago maydis homologous protein as a template. our results indicate that,in spite of the evolutionary divergence between both organisms,the structure of these two rpa-1 proteins seems to be highly conserved. conclusion: the lbrpa-1 protein is a ssdna binding protein,but also it shows affinity in vitro for the hsp70 mrna; this finding supports a possible in vivo role in the hsp70 mrna metabolism. on the other hand,the three dimensional model of leishmania rpa-1 serves as a starting point for both functional analysis and its exploration as a chemotherapeutic target to combat leishmaniasis. © 2014 nocua et al.