Expression, Functionality, and Localization of Apurinic/<scp>A</scp>pyrimidinic Endonucleases in Replicative and Non‐<scp>R</scp>eplicative Forms of <i>Trypanosoma cruzi</i>

Sepúlveda, Soledad; Valenzuela, Lucía; Ponce, Iván; Sierra, Soledad; Ramírez, Santiago R.; Rojas, Verónica; Kemmerling, Ulrike; Galanti, Norbel; Cabrera, Gonzalo

doi:10.1002/jcb.24675

Cited by 16 publications

(55 citation statements)

References 84 publications

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“…The absence of an inhibitory in vivo effect suggests that endogenous TcAP1 overcomes the TcAP1DN inhibitory activity. This is consistent with our previous results showing that TcAP1 is active and present as a constitutive enzyme in the three cellular forms of the parasite …”

Section: Discussionsupporting

confidence: 94%

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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Section: Discussionsupporting

confidence: 94%

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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“…Notably, APE2’s 3′–5′ exonuclease activity has been demonstrated and characterized in vitro from experimental model organisms including Arabidopsis thaliana , Trypanosoma cruzi , Ciona intestinalis , Saccharomyces cerevisiae , Schizosaccharomyces pombe , Xenopus laevis , and Homo sapiens [19,36,39,40,41,42,43], suggesting that the role of APE2’s 3′–5′ exonuclease activity in SSB end resection is highly conserved during evolution. APE2 3′–5′ exonuclease activity is important for the removal of 3′-blocked termini to repair DNA lesions from hydrogen peroxide treatment in Saccharomyces cerevisiae [38].…”

Section: Molecular Mechanism Of Ssb End Resectionmentioning

confidence: 99%

Single-Strand Break End Resection in Genome Integrity: Mechanism and Regulation by APE2

Hossain

Lin

Shan

2018

IJMS

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DNA single-strand breaks (SSBs) occur more than 10,000 times per mammalian cell each day, representing the most common type of DNA damage. Unrepaired SSBs compromise DNA replication and transcription programs, leading to genome instability. Unrepaired SSBs are associated with diseases such as cancer and neurodegenerative disorders. Although canonical SSB repair pathway is activated to repair most SSBs, it remains unclear whether and how unrepaired SSBs are sensed and signaled. In this review, we propose a new concept of SSB end resection for genome integrity. We propose a four-step mechanism of SSB end resection: SSB end sensing and processing, as well as initiation, continuation, and termination of SSB end resection. We also compare different mechanisms of SSB end resection and DSB end resection in DNA repair and DNA damage response (DDR) pathways. We further discuss how SSB end resection contributes to SSB signaling and repair. We focus on the mechanism and regulation by APE2 in SSB end resection in genome integrity. Finally, we identify areas of future study that may help us gain further mechanistic insight into the process of SSB end resection. Overall, this review provides the first comprehensive perspective on SSB end resection in genome integrity.

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“…In previous work, by site‐directed mutagenesis we have modified the nucleotide sequence that encodes the human APE1 endonuclease protein to obtain a putative negative dominant form …”

Section: Resultsmentioning

confidence: 99%

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival

Valenzuela

Sepúlveda

Ramírez-Toloza

et al. 2019

J of Cellular Biochemistry

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Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)‐specific parasite TcAP1 endonuclease is involved in the resistance to H2O2. However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H2O2. Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas’ disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell‐derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN‐GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell‐derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN‐GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H2O2 concentrations. No such effect was evident in expressing hAPE1DN‐GFP cell‐derived trypomastigotes. Although the viability of both wild‐type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H2O2 exposure than cell‐derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.

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

Cited by 16 publications

References 84 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

Single-Strand Break End Resection in Genome Integrity: Mechanism and Regulation by APE2

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival

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