Abstract
Trypanosoma cruzi, the etiological agent of Chagas’ disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing.
Highlights
Trypanosoma cruzi (T. cruzi), a hemoflagellate protozoan parasite, is the etiological agent of Chagas’ disease, an endemic pathology in Latin America [1]
An orthologous DNA coding sequence for a TcNTH1 DNA glycosylase protein (756 bp, GeneBank accession number 71412347) was detected in the T. cruzi genome corresponding to Homo sapiens NTH1 (GeneBank accession number U79718) and E. coli Endo III (GeneBank accession number AIFA01000047, locus tag ECDEC2A_2057)
Key catalytic amino acids specific of all DNA glycosylases [40,45] and cysteines that bind (4Fe-4S)2+ [40] present in E. coli, G. stearothermophilus, H. sapiens and L. infantum are fully conserved in the amino acid sequences of the enzymes depicted in Fig 1A, including TcNTH1
Summary
Trypanosoma cruzi (T. cruzi), a hemoflagellate protozoan parasite, is the etiological agent of Chagas’ disease, an endemic pathology in Latin America [1]. Twenty eight million people are at risk of exposure to infection with an estimated 6–7 million chronic cases in 21 endemic countries and 20.000 deaths per year. It is the parasitic disease with greater economic burden in America due to its long chronicity [2]. Chagas’ disease is transmitted by T. cruzi infected triatomine insects that upon feeding on mammal blood, deposit feces with metacyclic infective trypomastigotes. Epimastigotes are submitted to oxidative species during hemoglobin catabolism; those that survive multiply and move to the insect hindgut where they differentiate into infective metacyclic trypomastigotes [11,15,16,17]. The parasite is able to resist oxidative stress at different stages of its life cycle, suggesting a high efficiency of its DNA repair machinery
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