title: Leishmania braziliensis replication protein A subunit 1: molecular modelling, protein expression and analysis of its affinity for both DNA and RNA.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratorio de Parasitología Molecular, Facultad de Ciencias, Pontificia Universidad Javeriana, Carrera 7 No 43-82, Edificio 50, Laboratorio 113, Bogotá, Colombia. pnocua@javeriana.edu.co.
authors.forename: Paola A
authors.initials: PA
authors.lastname: Nocua
authors.affiliation: Laboratorio de Parasitología Molecular, Facultad de Ciencias, Pontificia Universidad Javeriana, Carrera 7 No 43-82, Edificio 50, Laboratorio 113, Bogotá, Colombia. cramirezbiologo@gmail.com.
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Ramirez
authors.affiliation: Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia. gesbarreto@gmail.com.
authors.forename: George E
authors.initials: GE
authors.lastname: Barreto
authors.affiliation: Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia. janneth.gonzalez@gmail.com.
authors.forename: Janneth
authors.initials: J
authors.lastname: González
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain. jmrequena@cbm.uam.es.
authors.forename: José M
authors.initials: JM
authors.lastname: Requena
authors.affiliation: Laboratorio de Parasitología Molecular, Facultad de Ciencias, Pontificia Universidad Javeriana, Carrera 7 No 43-82, Edificio 50, Laboratorio 113, Bogotá, Colombia. cpuerta@javeriana.edu.co.
authors.forename: Concepción J
authors.initials: CJ
authors.lastname: Puerta
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050884:HSP72 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007892:Leishmania braziliensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016773:Leishmaniasis, Cutaneous / Q000235:genetics / Q000378:metabolism* / Q000469:parasitology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D004268:DNA-Binding Proteins; D050884:HSP72 Heat-Shock Proteins; D021122:Protein Subunits; D015800:Protozoan Proteins; D051716:Replication Protein A; D012313:RNA; D004247:DNA