Apurinic/apyrimidinic endonuclease genes from the Trypanosomatidae Leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli
J. Perez et al., Apurinic/apyrimidinic endonuclease genes from the Trypanosomatidae Leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli, NUCL ACID R, 27(3), 1999, pp. 771-777
Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagen
ic and must be corrected to preserve genetic integrity. We have isolated cD
NAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capabl
e of complementing the deficiency of exonuclease III and dUTPase in the Esc
herichia coil mutant BW286. This double mutant is non-viable at 37 degrees
C due to an accumulation of non-repaired sites following excision of uracil
from DNA. The genes were expressed as beta-galactosidase-AP endonuclease f
usion proteins and as such are active in repair of AP sites in E.coli. The
Trypanosoma and Leishmania sequences have unique N-terminl containing seque
nces that correspond to probable nuclear transport signals, while the C-ter
minal domains exhibit pronounced similarity to exonuclease Ill. The L.major
gene was overexpressed as a histidine-tagged protein and recombinant enzym
e exhibited endonuclease activity on AP DNA in vitro. Furthermore, expressi
on of the enzymes in AP endonuclease-deficient E.coli mutants conferred sig
nificant resistance to killing by methylmethane sulphonate and peroxides. T
his study constitutes one of the first descriptions of DNA repair enzymes i
n these pathogenic organisms where oxidative stress is an important mechani
sm of both drug mediated and intracellular killing.