Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

Ormeño, Fernando; Barrientos, Camila; Ramírez, Santiago R.; Ponce, Iván; Valenzuela, Lucía; Sepúlveda, Soledad; Bitar, Mainá; Kemmerling, Ulrike; Machado, Carlos Renato; Cabrera, Gonzalo; Galanti, Norbel

doi:10.1371/journal.pone.0157270

Cited by 6 publications

(14 citation statements)

References 94 publications

(126 reference statements)

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“…While lipids and proteins may be re-synthesized DNA damage have to be repaired [10][11][12] mainly by the DNA Base Excision Repair (BER) pathway. [13][14][15][16] BER is a highly conserved pathway in eukaryotes that begins with the recognition of the oxidized base by one of many DNA glycosylases, which remove the damaged base, generating an AP site that is recognized by an apurinic/apyrimidinic endonuclease (AP endonuclease). This enzyme cleaves the DNA strand resulting in the formation of a one-nucleotide gap flanked by 3′-hydroxyl and 5′-deoxyribosephosphate (5′ dRP) ends.…”

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confidence: 99%

The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage

Valenzuela

Sepúlveda

Ponce

et al. 2018

J of Cellular Biochemistry

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Trypanosoma cruzi, the causative agent of Chagas' disease survives to DNA damage generated by ROS/RNS inside to their different hosts. In recent eukaryotes, oxidative DNA damage is repaired mainly by the Base Excision Repair (BER) pathway, being essential the apurinic/apyrimidinic endonuclease activity. Using a pTREX-gfp vector, the nucleotide sequence that encodes T. cruzi AP endonuclease TcAP1 (orthologue of human APE1) and a putative TcAP1 dominant negative (TcAP1DN), were transfectedand expressed in T. cruzi epimastigotes. TcAP1-GFP and TcAP1DN-GFP were expressed in those modified epimastigotes and found in the parasite nucleus. The endonucleases were purified under native conditions and the AP endonuclease activity was evaluated. While TcAP1 presents the expected AP endonuclease activity TcAP1DN does not. Moreover, TcAP1DN partially inhibits in vitro TcAP1 enzymatic activity. Transfected epimastigotes expressing TcAP1-GFP and TcAP1DN-GFP were differentiated to infective trypomastigotes. The infective parasites maintained both proteins (TcAP1-GFP and TcAP1DN-GFP) in the nucleus. The overexpression of TcAP1-GFP in epimastigotes and trypomastigotes increases the viability of both parasite forms when exposed to oxidative stress while the expression of TcAP1DN-GFP did not show any in vivo inhibitory effect, suggesting that endogenous TcAP1 constitutive expression overcomes the TcAP1DN inhibitory activity. Our results show that TcAP1 is important for trypomastigote survival under oxidative conditions similar to those found in infected mammalian cells, then increasing its permanence in the infected cells and the possibility of development of Chagas disease.

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The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage

Valenzuela

Sepúlveda

Ponce

et al. 2018

J of Cellular Biochemistry

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“…TcFEN1 is found in aggregates in the nucleoplasm. In previous work, we have shown that using pTREXgfp control vector, the expression of the green fluorescent protein remains in the cytoplasm [Sep ulveda et al, 2014;Ormeño et al, 2016]. The subcellular localization of TcFEN1 was maintained when the parasites were treated with H 2 O 2 (not shown).…”

Section: Expression and Activity Of Purified Recombinant His-tcfen1-gmentioning

confidence: 83%

“…Transfected epimastigotes over‐expressing HIS‐TcFEN1‐GFP maintained in the exponential phase of growth were exposed to a sustained H 2 O 2 production using the glucose/glucose oxidase system [Sepúlveda et al, ; Ormeño et al, ] for 2 (Fig. A) and (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We were not able to find the fusion TcFEN1‐GFP enzyme in the parasite kinetoplast; this is not an unexpected result taking in consideration that TcOrc1/Cdc and TcPCNA proteins are restrained to the nucleus [Godoy et al, ; Calderano et al, ]. Similarly, DNA base excision repair enzymes TcAPE1, TcAPE2, and TcNth1 are also located in the parasite nucleus only [Sepúlveda et al, ; Ormeño et al, ] while TcOGG1 (another DNA BER enzyme) is present in both compartments [Machado‐Silva et al, ].…”

Section: Discussionmentioning

confidence: 97%

“…Both, replicative and non-replicative, infective forms of the parasite are exposed to oxygen and nitrogen reactive species (ROS/ RNS) in its insect and mammal hosts, damaging its DNA [Cardoni et al, 1997;Graca-Souza et al, 2006;Piacenza et al, 2009;Cabrera et al, 2011]. In mammals, a DNA base excision repair mechanism is well established [Dianov et al, 1998;Robertson et al, 2009] and the parasite is able to repair oxidative DNA damage basically using the same pathway [Cabrera et al, 2011;Sep ulveda et al, 2014;Machado-Silva et al, 2016;Ormeño et al, 2016]. Thus, the infection is not fully eliminated and the vertebrate host will serve as a parasite reservoir, establishing a chronic infection [Peluffo et al, 2004].…”

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A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival

Ponce

Aldunate

Valenzuela

et al. 2016

J of Cellular Biochemistry

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FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H O . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc.

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Analysis of Base Excision and Single-Strand Break Repair Activities in Trypanosomatid Extracts

Kania

Ginger²,

Allinson

2020

Methods in Molecular Biology

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Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

Cited by 6 publications

References 94 publications

The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage

The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage

A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival

Analysis of Base Excision and Single-Strand Break Repair Activities in Trypanosomatid Extracts

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