Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

• Published in: Nucleic acids research Vol. 29; no. 7; pp. 1549 - 1555
• Main Authors: Fárez-Vidal, M E, Gallego, C, Ruiz-Pérez, L M, González-Pacanowska, D
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01-04-2001; Oxford University Press
• Subjects: Amino Acid Sequence; Animals; AP endonuclease; Blotting, Western; Carbon-Oxygen Lyases - metabolism; Chromosome Mapping; Deoxyribonuclease IV (Phage T4-Induced); DNA Glycosylases; DNA, Complementary - chemistry; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; DNA-(Apurinic or Apyrimidinic Site) Lyase; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Exodeoxyribonucleases - metabolism; exonuclease III; Leishmania major; Molecular Sequence Data; N-Glycosyl Hydrolases - genetics; N-Glycosyl Hydrolases - metabolism; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Trypanosoma cruzi; Trypanosoma cruzi - enzymology; Trypanosoma cruzi - genetics; Uracil-DNA Glycosidase; uracil-DNA glycosylase
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/29.7.1549

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas' disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of approximately 1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.