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

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Ch agas' disease. We have isolated a full-length cDNA encoding uracil-DNA glyc osylase (UDGase), a key enzyme involved in DNA repair, from this organism, The deduced protein sequence is highly conserved at the C-terminus of the m olecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variab le, The gene is single copy and is located on a chromosome of similar to1.9 Mb, A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existenc e of a single UDGase species in parasite extracts, Using a specific ethidiu m bromide fluorescence assay, recombinant T.cruzi UDGase was shown to speci fically excise uracil from DNA, The addition of both Leishmania major AP en donuclease and exonuclease ill, 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 e nzymes.