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

Abstract

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.

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…In humans, DNA synthesis is believed to be carried out by DNA polymerase eta (141). The cloning and characterization of T. cruzi DNA polymerase eta were reported (142). Purified polymerase eta promoted DNA synthesis in primer extension assays and bypassed oxidative DNA lesions such as 8-oxoguanine.…”

“…However, it complements the UV sensitivity of polymerase eta-deficient yeast cells. The low fidelity of polymerase eta might provide the parasite with adaptive mutations, allowing it to escape from host immune surveillance (142). It remains to be seen whether T. cruzi polymerase eta also extends D-loops.…”

Overview of DNA Repair inTrypanosoma cruzi, Trypanosoma brucei,andLeishmania major

“…This could be related to the peculiar constitutive transcription of Tritryps. In fact, overexpression of T. cruzi DNA polymerase η (Pol η ), involved in the translesion synthesis of pirimidine dimers, and overexpression or haploinsufficiency of RAD51, a key protein in HR, do not confer any protection against UV irradiation, which could suggest that the UV-induced lesions are fully repaired before the cell enters the S-phase ([39], Passos-Silva et al, submitted). In addition, results obtained by our group show that T. cruzi repairs cisplatin-induced lesions at an extremely high rate, with total lesion removal in less than an hour (Rajão, unpublished data).…”

“…Pol η from T. cruzi has been characterized in vitro and in vivo [39]. TcPol η is able to complement yeast Rad30 mutant (Pol η -null mutant), increasing yeast resistance to UV radiation, which indicates that Pol η is able to bypass UV lesions.…”

“…In contrast to the result seen in yeast, parasites overexpressing this nuclear polymerase do not show a higher resistance to UV radiation. The lack of conferred resistance might be related to the number of lesions remaining during S phase because it is possible that the majority of UV lesions would be repaired by TCR-NER prior to DNA replication, as the majority of the protein-coding genes are constitutively transcribed in this organism [39]. …”