title: Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form.
abstract: Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.
authors.affiliation: Programa de Biología Celular y Molecular, Facultad de Medicina, Instituto de Ciencias Biomédicas (ICBM), Universidad de Chile, Santiago, Chile.
authors.forename: Edio
authors.initials: E
authors.lastname: Maldonado
authors.forename: Diego A
authors.initials: DA
authors.lastname: Rojas
authors.forename: Sandra
authors.initials: S
authors.lastname: Moreira-Ramos
authors.forename: Fabiola
authors.initials: F
authors.lastname: Urbina
authors.forename: Vicente J
authors.initials: VJ
authors.lastname: Miralles
authors.forename: Aldo
authors.initials: A
authors.lastname: Solari
authors.forename: Juan
authors.initials: J
authors.lastname: Venegas
mesh_terms: D014355:Chagas Disease / Q000469:parasitology*; D019951:DNA Polymerase beta / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000737:chemistry / Q000235:genetics; D054306:Dideoxynucleotides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D010766:Phosphorylation; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013942:Thymine Nucleotides / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
chemical_list: D018105:DNA, Kinetoplast; D054306:Dideoxynucleotides; D004791:Enzyme Inhibitors; D013942:Thymine Nucleotides; D019951:DNA Polymerase beta; C017216:2',3'-dideoxythymidine triphosphate; D005033:Ethylmaleimide