Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase.
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.
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Fernando
F
Ormeño
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Camila
C
Barrientos
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Santiago
S
Ramirez
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Iván
I
Ponce
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Lucía
L
Valenzuela
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Sofía
S
Sepúlveda
Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Mainá
M
Bitar
Programa de Anatomía y Biología del Desarrollo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Ulrike
U
Kemmerling
Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Carlos Renato
CR
Machado
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Gonzalo
G
Cabrera
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Norbel
N
Galanti
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D014355:Chagas Disease / Q000469:parasitology*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D005786:Gene Expression Regulation; D006801:Humans; D008958:Models, Molecular; D018384:Oxidative Stress; D011487:Protein Conformation; D051381:Rats; D016415:Sequence Alignment; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*
C029389:thymine glycol; D045647:DNA Glycosylases; D013941:Thymine