DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions

Abstract

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.

Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

“…Importantly, although there are other recombinant Tcpolß described in the literature; they are from the CL Brener clone of the TcVI lineage which were expressed as fusion peptides bound to maltose-binding protein (Lopes et al 2008;Schamber-Reis et al 2012). As we mentioned in a previous paper, despite the similarity of the TcI polß Miranda clone to the CL Brener clone, they are not identical (Venegas et al 2009).…”

“…An important consideration in the study of this parasite is the particular biological and biochemical characteristics of its single and peculiar mitochondrion (kinetoplast), which is composed of a complex network of minicircles and maxicircles of DNA that are replicated by a unique mechanism (Ferguson et al 1991). According to several studies, the kinetoplast plays a critical role in parasite locomotion, suggesting that its biochemical and molecular mechanisms would be favored as specific chemotherapeutic targets (Schamber-Reis et al 2012). In kinetoplast replication, the DNA polymerase beta (polß) has a key function in DNA repair (Torri and Englund 1995;Saxowsky et al 2003;Schamber-Reis et al 2012).…”

“…According to several studies, the kinetoplast plays a critical role in parasite locomotion, suggesting that its biochemical and molecular mechanisms would be favored as specific chemotherapeutic targets (Schamber-Reis et al 2012). In kinetoplast replication, the DNA polymerase beta (polß) has a key function in DNA repair (Torri and Englund 1995;Saxowsky et al 2003;Schamber-Reis et al 2012). T. cruzi has a complex life cycle in both the vertebrate and the invertebrate hosts, where different parasitic forms exist, the epimastigote, amastigote, and trypomastigote, which are exposed to different oxidative stresses.…”

“…At present, the main lineage TCI is called TcI, and DTU 2a, DTU 2b, DTU 2c, DTU 2d, and DTU 2e are called TcIV, TcII, TcIII, TcV, and TcVI, respectively (Zingales et al 2012). The T. cruzi CL Brener clone used in previous studies of T. cruzi polß belongs to the hybrid lineage TcVI (Schamber-Reis et al 2012;Lopes et al 2008). It is noteworthy that the genome sequencing of this clone confirmed not only that this parasite is predominantly diploid but also that the nuclear genome is formed by genes similar to those described for the T. cruzi Esmeraldo clone belonging to the TcII lineage (Esmeraldo haplotype) and similar to those described for TcIII clones (non-Esmeraldo haplotype) (ElSayed et al 2005a).…”

“…A later report based on transfection of T. cruzi parasites with an expression vector containing the full-length polß gene from the T. cruzi CL Brener clone demonstrated that parasites overexpressing polß showed increased survival in a range of hydrogen peroxide concentrations, suggesting that polß contributed to the repair of oxidative lesions of kDNA and improved the efficiency of base excision repair (Schamber-Reis et al 2012). However, on those in vitro studies with the fusion peptide of MBP-T. cruzi polymerase beta (Tcpolß), the recombinant enzyme could not perform base excision repair or overcome DNA lesion with 8-oxodG (Lopes et al 2008).…”