Apurinic/apyrimidinic endonuclease genes from the Trypanosomatidae Leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli

• Published in: Nucleic acids research 1999-02, Vol.27 (3), p.771-777
• Main Authors: Pérez, Juana, Gallego, Cláribel, Bernier-Villamor, Víctor, Camacho, Ana, González-Pacanowska, Dolores, Ruiz-Pérez, Luis M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Carbon-Oxygen Lyases - genetics; Deoxyribonuclease IV (Phage T4-Induced); DNA Repair - genetics; DNA-(Apurinic or Apyrimidinic Site) Lyase; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli Proteins; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; Genetic Complementation Test; Leishmania major; Leishmania major - enzymology; Leishmania major - genetics; Methyl Methanesulfonate - pharmacology; Molecular Sequence Data; Mutagenesis; Mutagens - pharmacology; Oxidation-Reduction; Peroxides - pharmacology; Sequence Alignment; Trypanosoma cruzi; Trypanosoma cruzi - enzymology; Trypanosoma cruzi - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/27.3.771

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37°C due to an accumulation of nonrepaired sites following excision of uracil from DNA. The genes were expressed as β-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E.coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drugmediated and intracellular killing.