Functional Characterization of 8-Oxoguanine DNA Glycosylase of Trypanosoma cruzi

Abstract

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1−/− (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H2O2). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H2O2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

Expression, Functionality, and Localization of Apurinic/Apyrimidinic Endonucleases in Replicative and Non‐Replicative Forms of Trypanosoma cruzi

“…BER is an evolutionarily conserved process for maintaining nuclear and mitochondrial genomic integrity by eliminating oxidized, alkylated or mismatched bases that are generated endogenously or induced by ROS/RNS [Mandavilli et al, ; Hegde et al, ]. To date, the presence and activity of several enzymes of the BER pathway, such as UDGase (Uracil‐DNA glycosylase), PARP and 8‐oxoguanine DNA glycosylase have been studied in T. cruzi [Farez‐Vidal et al, ; Pena‐Diaz et al, ; Fernandez Villamil et al, ; Furtado et al, ]. Furthermore, the APE1 gene (TcAP1) has been described in this parasite; expression of this gene confers resistance to oxidant and alkylating agents when probed in hypersensitive E. coli , deficient in exonuclease III enzyme [Perez et al, ].…”

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…The expression of TcOGG1 complemented an Ogg1-defective Saccharomyces cerevisiae strain when assayed for spontaneous mutation frequency. Moreover, TcOGG1 reduced the levels of 8-oxoG in the nucleus and in the mitochondrion of T. cruzi (52).…”

Distinct Phenotypes Caused by Mutation of MSH2 in Trypanosome Insect and Mammalian Life Cycle Forms Are Associated with Parasite Adaptation to Oxidative Stress

“…cruzi strains and mutants for different DNA repair proteins [37, 49, 50]. Briefly, 20 μL aliquots of cell suspensions were distributed on a glass slide coated with poly-lysine (Sigma-Aldrich) and left to adhere for 5–10 min, after which cells were fixed with ice-cold methanol for 20 min at -20°C.…”