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

Abstract

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 Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…AP endonuclease 1 (APN1) is the primary type in budding yeast and is homologous to Escherichia coli endonuclease IV, while APN2 is related to exonuclease III. In 1999, Perez et al provided the first report on BER enzymes by identifying AP endonuclease in L. major and T. cruzi (38). The L. major apurinic/apyrimidinic endonuclease (LmAP) belongs to the APE1/ExoIII family ( Table 1).…”

Crystal Structure and DNA Repair Activities of the AP Endonuclease from Leishmania major

“…Its expression in E. coli hypersensitive mutants was shown to confer resistance to oxidative and alkylating agents and the purified histidine-tagged enzyme exhibited AP endonuclease activity in vitro. 23 To characterize further its enzymatic activities, the His-LMAP protein (∼56 kDa) was overexpressed in E. coli BL21 (DE3) and purified to over 95% purity by histidine-affinity and gel filtration chromatography.…”

“…It shows AP endonuclease activity and its expression in AP endonuclease-deficient E. coli mutants confers protection against oxidative agents and resistance to killing by alkylating agents. 23 Protozoan parasites are highly sensitive to macrophage-generated oxidative stress 24 but their prevalence indicates that they possess mechanisms to counteract the deleterious effects of free radical attack. Protozoan specific differences in the mechanisms of oxidant defense may be able to be exploited in the development of new therapies.…”

Characterization of class II apurinic/apyrimidinic endonuclease activities in the human malaria parasite, Plasmodium falciparum

“…The Trypanosoma and Leishmania sequences had unique N-terminicontaining sequences corresponding to nuclear import signals, whereas the C-terminal domains exhibited pronounced similarity to ExoIII. The expression in i o of the AP endonuclease from T. cruzi conferred viability by complementation to E. coli ExoIII deletion mutants [36]. The presence of a Mg# + -dependent class II AP endonuclease activity in the P. falciparum lysate suggests that this might be an ExoIII (functional) homologue.…”

“…A recent investigation of the AP endonucleases in the related Trypanosomatidae Leishmania major and Trypanosoma cruzi identified the gene for a class II AP endonuclease [36,37]. The Trypanosoma and Leishmania sequences had unique N-terminicontaining sequences corresponding to nuclear import signals, whereas the C-terminal domains exhibited pronounced similarity to ExoIII.…”