title: A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.
abstract: FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H2O2. The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Ivan
authors.initials: I
authors.lastname: Ponce
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Carmen
authors.initials: C
authors.lastname: Aldunate
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Lucia
authors.initials: L
authors.lastname: Valenzuela
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Sofia
authors.initials: S
authors.lastname: Sepúlveda
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Gilda
authors.initials: G
authors.lastname: Garrido
authors.affiliation: Programa de Anatomía y Biología del Desarrollo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kemmerling
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Cabrera
authors.affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
authors.forename: Norbel
authors.initials: N
authors.lastname: Galanti
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019295:Computational Biology; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D006861:Hydrogen Peroxide; D045585:Flap Endonucleases
keywords: DNA REPAIR; PARASITE PROLIFERATION AND SURVIVAL; TRYPANOSOMA CRUZI; TcFEN1