Pérez J, Gallego C, Bernier-Villamor V, Camacho A, González-Pacanowska D, Ruiz-Pérez L M
Instituto de Parasitología y Biomedicina 'López-Neyra', Consejo Superior de Investigaciones Científicas,C/ Ventanilla 11, 18001-Granada, Spain.
Nucleic Acids Res. 1999 Feb 1;27(3):771-7. 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 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-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 drug-mediated and intracellular killing.
DNA中的脱嘌呤/脱嘧啶（AP）位点被认为具有高度致突变性，必须加以修复以保持遗传完整性。我们从锥虫科的硕大利什曼原虫和克氏锥虫中分离出了cDNA，它们能够弥补大肠杆菌突变体BW286中外切核酸酶III和dUTPase的缺陷。由于从DNA中切除尿嘧啶后未修复位点的积累，这个双突变体在37摄氏度时无法存活。这些基因表达为β-半乳糖苷酶-AP内切核酸酶融合蛋白，因此在大肠杆菌中对AP位点的修复具有活性。锥虫和利什曼原虫的序列具有独特的N端，其中包含与可能的核转运信号相对应的序列，而C端结构域与外切核酸酶III表现出明显的相似性。硕大利什曼原虫基因作为组氨酸标签蛋白被过度表达，重组酶在体外对AP DNA表现出内切核酸酶活性。此外，在缺乏AP内切核酸酶的大肠杆菌突变体中表达这些酶，可使其对甲基磺酸甲酯和过氧化物的杀伤具有显著抗性。这项研究首次描述了这些致病生物中的DNA修复酶，在这些生物中，氧化应激是药物介导杀伤和细胞内杀伤的重要机制。
