Me. Farez-vidal et al., Characterization of uracil-DNA glycosylase activity from Trypanosoma cruziand its stimulation by AP endonuclease, NUCL ACID R, 29(7), 2001, pp. 1549-1555
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.