Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Lopes, Débora de Oliveira; Schamber-Reis, Bruno Luiz Fonseca; Régis-da-Silva, Carlos Gustavo; Rajão, Matheus Andrade; DaRocha, Wanderson D.; Macedo, Andréa Mara; Franco, Glória Regina; Nardelli, Sheila Cristina; Schenkman, Sérgio; Hoffmann, Jean-Sébastien; Cazaux, Christophe; Pena, Sérgio Danilo Junho; Teixeira, Santuza Maria Ribeiro; Machado, Carlos Renato

doi:10.1016/j.dnarep.2008.07.018

Cited by 26 publications

(43 citation statements)

References 19 publications

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“…All of the deduced amino acid sequences for these polßs have putative mitochondrial presequences. These genomic studies confirmed the previous facts revealed from the analysis of T. brucei, showing that the paralog sequence named DNA polß-PAK is found near the polß gene (Lopes et al 2008;El-Sayed et al 2005a). In addition to polß and polß-PAK, five other mitochondrial DNA polymerases (pols) have been identified in trypanosomatids (Klingbeil et al 2002;Chandler et al 2008;Rajao et al 2009;Bruhn et al 2010).…”

Section: Introductionsupporting

confidence: 89%

“…The polß gene from the T. cruzi TcVI lineage CL Brener clone (Lopes et al 2008) was expressed as fusion peptides of polß with maltose-binding protein (MBP); this study demonstrated that the enzyme has DNA polymerase and dRPlyase activities, and it is inhibited by NaCl (0-100 mM) and has an optimal pH of about 8.0 (Lopes et al 2008). A later report based on transfection of T. cruzi parasites with an expression vector containing the full-length polß gene from the T. cruzi CL Brener clone demonstrated that parasites overexpressing polß showed increased survival in a range of hydrogen peroxide concentrations, suggesting that polß contributed to the repair of oxidative lesions of kDNA and improved the efficiency of base excision repair (Schamber-Reis et al 2012).…”

Section: Introductionmentioning

confidence: 99%

“…At present, the main lineage TCI is called TcI, and DTU 2a, DTU 2b, DTU 2c, DTU 2d, and DTU 2e are called TcIV, TcII, TcIII, TcV, and TcVI, respectively (Zingales et al 2012). The T. cruzi CL Brener clone used in previous studies of T. cruzi polß belongs to the hybrid lineage TcVI (Schamber-Reis et al 2012;Lopes et al 2008). It is noteworthy that the genome sequencing of this clone confirmed not only that this parasite is predominantly diploid but also that the nuclear genome is formed by genes similar to those described for the T. cruzi Esmeraldo clone belonging to the TcII lineage (Esmeraldo haplotype) and similar to those described for TcIII clones (non-Esmeraldo haplotype) (ElSayed et al 2005a).…”

Section: Introductionmentioning

confidence: 99%

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Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

et al. 2015

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Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.

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Section: Introductionsupporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

et al. 2015

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“…DOI 10.1002/em TcPolh sequence showed high identity in the N-terminal region with the five conserved motifs of Y family polymerases [Johnson et al, 1999b;Masutani et al, 1999], the amino acid residues important for the catalytic activity [Glick et al, 2001] and a conserved PCNA biding motif that allows TcPolh-PCNA interaction, which is essential for the in vivo activity of Polh [Haracska et al, 2001a]. TcPolh is the first polymerase found to be localized in the nucleus, considering that DNA polymerases b, b-PAK, and j are located in the mitochondria of this organism [Lopes et al, 2008].…”

Section: Discussionmentioning

confidence: 98%

“…TcPolh protein was able to insert a nucleotide in front of the lesion (12 position). However, TcPolh was less efficient in bypassing this lesion than TcPolb-PAK, a DNA translesion protein present in T. cruzi mitochondria [Lopes et al, 2008]. In contrast to TcPolh and TcPolb-PAK, TcPolb was unable to bypass this lesion and stopped the polymerization just before the lesion (Fig.…”

Section: Treatment Of T Cruzi Tcpolg-overexpressing Parasites With Hmentioning

confidence: 96%

Cloning and characterization of DNA polymerase η from Trypanosoma cruzi: Roles for translesion bypass of oxidative damage

Moura

Schamber-Reis

Silva

et al. 2009

Environ and Mol Mutagen

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We report the cloning and characterization of the DNA polymerase eta gene from Trypanosoma cruzi (TcPoleta), the causative agent of Chagas disease. This protein, which can bypass cyclobutane pyrimidine dimers, contains motifs that are conserved between Y family polymerases. In vitro assays showed that the recombinant protein is capable of synthesizing DNA in undamaged primer-templates. Intriguingly, T. cruzi overexpressing TcPoleta does not increase its resistance to UV-light (with or without caffeine) or cisplatin, despite the ability of the protein to enhance UV resistance in a RAD30 mutant of Saccharomyces cerevisiae. Parasites overexpressing TcPoleta are also unable to restore growth after treatment with zeocin or gamma irradiation. T. cruzi overexpressing TcPoleta are more resistant to treatment with hydrogen peroxide (H(2)O(2)) compared to nontransfected cells. The observed H(2)O(2) resistance could be associated with its ability to bypass 8-oxoguanine lesions in vitro. The results presented here suggest that TcPoleta is able to bypass UV and oxidative lesions. However the overexpression of the gene only interferes in response to oxidative lesions, possibly due to the presence of these lesions during the S phase.

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Unveiling Benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi

Rajão

Furtado

Alves

et al. 2013

Environ and Mol Mutagen

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Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.

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Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Cited by 26 publications

References 19 publications

Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

Cloning and characterization of DNA polymerase η from Trypanosoma cruzi: Roles for translesion bypass of oxidative damage

Unveiling Benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi

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