Matheus Andrade Rajão

Overview of DNA Repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major

A wide variety of DNA lesions arise due to environmental agents, normal cellular metabolism, or intrinsic weaknesses in the chemical bonds of DNA. Diverse cellular mechanisms have evolved to maintain genome stability, including mechanisms to repair damaged DNA, to avoid the incorporation of modified nucleotides, and to tolerate lesions (translesion synthesis). Studies of the mechanisms related to DNA metabolism in trypanosomatids have been very limited. Together with recent experimental studies, the genome sequencing of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, has revealed interesting features of the DNA repair mechanism in these protozoan parasites, which will be reviewed here.

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

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. Environ. Mol. Mutagen. 55:309–321, 2014.

Sm21.6 a novel EF-hand family protein member located on the surface of Schistosoma mansoni adult worm that failed to induce protection against challenge infection but reduced liver pathology.

Schistosomiasis continues to be a significant public health problem that affects 200 million people worldwide. This is one of the most important parasitic diseases, and one whose effective control is unlikely in the absence of a vaccine. In this study, we have isolated a cDNA clone encoding the Schistosoma mansoni Sm21.6 protein that has 45% and 44% identity with Sm22.6 and Sj21.7 EF-hand containing antigens, respectively. Confocal microscopy analysis revealed that Sm21.6 is a membrane-associated protein localized on the S. mansoni adult worm. Mouse immunization with rSm21.6 induced a mixed Th1/Th2 cytokine profile and no protection against infection. However, vaccination with rSm21.6 reduced by 28% of liver granuloma numbers, 21% of granuloma area and 34% of fibrosis. Finally, rSm21.6 was recognized by sera from individuals resistant to reinfection compared with patients susceptible to reinfection and this molecule should be further studied as potential biomarker for disease resistance. In conclusion, Sm21.6 is a new tegument protein from S. mansoni that plays an important role in reducing pathology induced by parasite infection.

DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate.

DNA polymerase kappa (Polκ) is a low‐fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y‐family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Polκ from the protozoan Trypanosoma cruzi (TcPolκ). The role of this TcPolκ copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N‐terminal extension which is present only in eukaryotic DinB members, but its C‐terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C2HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPolκ efficiently bypasses 8‐oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPolκ increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

Nucleoplasmic Calcium Buffering Sensitizes Human Squamous Cell Carcinoma to Anticancer Therapy

Background: Calcium (Ca2+) signaling within the nucleus is known to play a crucial role in cell proliferation. The aim of this study was to investigate whether nuclear Ca2+ buffering could improve the antitumor effect of X-rays therapy on Human Squamous Cell Carcinoma (HSCC). Methods: For these purpose, we developed an experimental protocol that simulated clinical radiotherapy and prevented bystander effects of irradiation. HSCC, A431 cell line, was submitted to 10Gy cumulative X-rays therapy alone (XR Cd 10Gy) or in association with the strategy that selectively buffer nuclear Ca 2+ (Ca 2+ n) signaling. Results: Upon Ca 2+ n buffering, A431 cell proliferation rate decreased significantly as compared to control. Cell cycle analysis showed that association of Ca2+ n buffering with XR Cd 10Gy increased the percentage of A431 cells at G 2 /M and did not increase nuclear/mitochondrial DNA damages. Nonetheless, Ca 2+ n buffering prevented the increase of the radioresistance-related biomarker ADAM-17 expression and EGFR activation induced by irradiation. Furthermore, the association therapy almost completely abolished cell survival fraction even using approximately half of the X-rays cumulative dose Conclusions: Nuclear Ca 2+ buffering sensitizes human squamous cell carcinoma to X - rays irradiation treatment.

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

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

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.

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

We report the cloning and characterization of the DNA polymerase η gene from Trypanosoma cruzi (TcPolη), 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 TcPolη 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 TcPolη are also unable to restore growth after treatment with zeocin or gamma irradiation. T. cruzi overexpressing TcPolη are more resistant to treatment with hydrogen peroxide (H2O2) compared to nontransfected cells. The observed H2O2 resistance could be associated with its ability to bypass 8‐oxoguanine lesions in vitro. The results presented here suggest that TcPolη 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. Environ. Mol. Mutagen. 2009.

Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

Background During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. Methodology/Principal Findings In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present. Conclusion Taken together, these results strongly support the hypothesis that SmZF1 acts as a transcription factor in S. mansoni.

Nucleotide excision repair in Trypanosoma brucei: specialization of transcription-coupled repair due to multigenic transcription

Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG‐NER), which is responsible for repair throughout genomes, and transcription‐coupled NER (TC‐NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC‐NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter‐strand cross‐link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.