Trypanosoma cruzi Contains a Single Detectable Uracil-DNA Glycosylase and Repairs Uracil Exclusively Via Short Patch Base Excision Repair

Peña-Dı́az, Javier; Akbari, Mansour; Sundheim, Ottar; Fárez-Vidal, María Esther; Andersen, Sonja; Sneve, Ragnhild; González-Pacanowska, Dolores; Krokan, Hans E.; Slupphaug, Geir

doi:10.1016/j.jmb.2004.07.043

Cited by 16 publications

(17 citation statements)

References 63 publications

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“…In addition, the activity was stimulated in the presence of AP endonuclease (44), similar to what was observed with the human enzymes (45). Moreover, a functional role for the enzyme was confirmed, since the expression of TcUNG in an E. coli ung mutant restored the WT phenotype (46). In T. brucei, ung-null mutant cell extracts did not perform excision of uracil in DNA, revealing the absence of a backup excision pathway when the specific glycosylase is not active (47).…”

Section: Dna Glycosylases and Ap Endonucleasessupporting

confidence: 72%

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

Genois

Paquet

Laffitte

et al. 2014

Microbiol Mol Biol Rev

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SUMMARY All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.

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Section: Dna Glycosylases and Ap Endonucleasessupporting

confidence: 72%

“…2A and B). It has been shown by using a covalently closed circular DNA (cccDNA) substrate containing a uracil that only SP-BER occurs in T. cruzi cell extracts (46). In the LP-BER pathway, the flap endonuclease 1 (FEN-1) cleaves within the apurinic/apyrimidinic (AP) site-terminated flap (48).…”

Section: Different Classes Of Ber Enzymesmentioning

confidence: 99%

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

Genois

Paquet

Laffitte

et al. 2014

Microbiol Mol Biol Rev

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“…This is the physiological temperature of human hosts of parasites. This optimal temperature was similar to that for UDG from other organisms except T. cruzi UDG, which showed optimal temperature at 45°C [24]. Both forms of PfUDG worked well in alkaline conditions (pH 7–9), so it is suggested to be one of the pH-dependent enzymes.…”

Section: Discussionsupporting

confidence: 62%

“…Both forms of PfUDG worked well in alkaline conditions (pH 7–9), so it is suggested to be one of the pH-dependent enzymes. This result relates to the optimal pH of UDG from other organisms [20,24,27]. …”

Section: Discussionmentioning

confidence: 99%

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Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target

Suksangpleng

Leartsakulpanich

Moonsom

et al. 2014

Malar J

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BackgroundBased on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target.MethodsInitially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests.ResultsThe native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino)uracil (IC50 of 16.75 μM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 μM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 μM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2 cells, with IC50 of > 160 μM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 μM.ConclusionsThe recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future.

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

Sepúlveda

Valenzuela

Ponce

et al. 2013

J of Cellular Biochemistry

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Trypanosoma cruzi is the etiological agent of Chagas disease. The parasite has to overcome oxidative damage by ROS/RNS all along its life cycle to survive and to establish a chronic infection. We propose that T. cruzi is able to survive, among other mechanisms of detoxification, by repair of its damaged DNA through activation of the DNA base excision repair (BER) pathway. BER is highly conserved in eukaryotes with apurinic/apirimidinic endonucleases (APEs) playing a fundamental role. Previous results showed that T. cruzi exposed to hydrogen peroxide and peroxinitrite significantly decreases its viability when co-incubated with methoxyamine, an AP endonuclease inhibitor. In this work the localization, expression and functionality of two T. cruzi APEs (TcAP1, Homo sapiens APE1 orthologous and TcAP2, orthologous to Homo sapiens APE2 and to Schizosaccaromyces pombe Apn2p) were determined. These enzymes are present and active in the two replicative parasite forms (epimastigotes and amastigotes) as well as in the non-replicative, infective trypomastigotes. TcAP1 and TcAP2 are located in the nucleus of epimastigotes and their expression is constitutive. Epimastigote AP endonucleases as well as recombinant TcAP1 and TcAP2 are inhibited by methoxyamine. Overexpression of TcAP1 increases epimastigotes viability when they are exposed to acute ROS/RNS attack. This protective effect is more evident when parasites are submitted to persistent ROS/RNS exposition, mimicking nature conditions. Our results confirm that the BER pathway is involved in T. cruzi resistance to DNA oxidative damage and points to the participation of DNA AP endonucleases in parasite survival.

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Trypanosoma cruzi Contains a Single Detectable Uracil-DNA Glycosylase and Repairs Uracil Exclusively Via Short Patch Base Excision Repair

Cited by 16 publications

References 63 publications

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

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

Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target

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

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