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

Abstract

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.

Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems

“…Furthermore, RFC and PCNA act as a loading platform for the flap endonuclease-1 (FEN-1) and DNA ligase I, which process Okazaki fragments 33 , 39 . FEN-1 in T. cruzi was recently shown to participate in DNA replication and repair [40] , but DNA ligase I has not yet been characterized in any trypanosomatid.…”

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

“…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.…”

DNA lesions and repair in trypanosomatids infection

“…The long-patch repair pathway also seems to be present in all forms of T. cruzi since a recent paper described the presence of Tc-Flap endonuclease 1 (TcFEN1), a homolog of human FEN1. In this work, TcFEN1 increased parasite resistance to oxidative stress when overexpressed; this protein deals with DNA intermediates containing a 5' flap, showing a protein specific to LP-BER in this parasite (Ponce et al, 2017).…”