br Materials and methods br

Materials and methods
Results
Discussion The GO system is an important mechanism for maintaining the integrity of genetic material in many eukaryotic organisms. This is demonstrated by the high conservation, across different evolutionary lineages, of the sequences of the proteins that compose this system (Jansson et al., 2010) and by the functional conservation of these enzymes (Takao et al., 1999, Bai et al., 2005, Ali et al., 2008). Trypanosoma cruzi CL Brener strain's genome annotation, in 2005, by El-Sayed and collaborators, showed that this parasite has homologs of GO system proteins. Given the importance of these enzymes in the maintenance of genome integrity, as well as their possible role in generating variability in T. cruzi, we decided to study these proteins. T. cruzi OGG1 (TcOGG1) and MTH (TcMTH) were previously described and characterized elsewhere (Furtado et al., 2012, Aguiar et al., 2013). In the present study, we characterized MYH from Trypanosoma cruzi (TcMYH). Given MutY/MYH sequence conservation between T. cruzi and other species (Fig. 1), heterologous complementation assays - specifically, spontaneous mutation assays - were carried out in E. coli to test the functions of TcMYH. Expression of TcMYH in E. coli mutY (BH980) reduced the mutation rate about twenty times, when compared to Plumbagin australia that do not express this gene, showing that TcMYH complemented BH980. This rifampicin spontaneous mutation assay is largely used and validated (Tajiri et al., 1995, Pope et al., 2008, Schaaper and Mathews, 2013), indicating that this result is strong evidence that TcMYH has MutY DNA glycosylase activity. E. coli was also employed for large-scale expression and purification of MBP- TcMYH, so that in vitro glycosylase tests could be performed. In vitro assays using synthetic oligonucleotides are commonly employed to characterize MYH orthologs (Takao et al., 1999, Bai et al., 2005, Ali et al., 2008; Fig. 2B). DNA breakage only in the presence of NaOH (Fig. 2C) confirms that TcMYH is a monofunctional DNA glycosylase. Therefore, after TcMYH activity it should be necessary to have activity of an AP endonuclease. There are also extra bands where TcMYH was added, probably due to spurious activity of TcMYH fused with MBP; however, this activity does not compromise the MutY glycosylase function of TcMYH, as the fragment indicating this function is only present when GO is opposite adenine, in the presence of NaOH. In contrast, the extra bands are present with or without NaOH addition, and even when GO is present opposite cytosine. MBP was purified using the same protocol as for TcMYH-MBP and it did not show the band related to MutY glycosylase function, neither the extra bands. Thereafter, T. cruzi epimastigotes overexpressing TcMYH were constructed (Fig. 3A) and TcMYH-overexpressor parasites showed to be more sensitive to hydrogen peroxide or glucose oxidase than control cells (Fig. 4 A and B). There are no reports in the literature of cytotoxic effects after overexpression of MutY, except in situations of high oxidative stress (Oka and Nakabeppu, 2011, Sheng et al., 2012). Cells that overexpress glycosylases are usually more sensitive to exogenous agents than control cells (Kaasen et al., 1986, Coquerelle et al., 1995, Frosina, 2000, Fishel et al., 2003, Rinne et al., 2005, Ondovcik et al., 2013). A similar result could be seen in our characterization of TcOGG1, where T. cruzi cells that overexpressed this protein were more sensitive to H2O2 than control cells (Furtado et al., 2012). Thus, our result indicates that a DNA glycosylase that removes oxidative lesions is being overexpressed in T. cruzi transfected with pROCK_TcMYH. This phenotype observed in glycosylase-overexpressing cells was attributed to impairment of the DNA BER pathway (Glassner et al., 1998, Posnick and Samson, 1999, Oka and Nakabeppu, 2011). Thus, as only the DNA glycosylase is being overexpressed and the other proteins that participate on repair do not accompany this elevated expression, intermediary lesions - such as AP sites - are produced but not completely repaired, giving rise to a sensitive phenotype. Therefore, accentuated death of TcMYH-overexpressing epimastigotes can be explained by insufficient processing of excess intermediary lesions (e.g. AP sites, SSBs, DSBs) generated by the high levels of TcMYH in a situation where substrate is not limiting. This hypothesis is corroborated by the AP assay result shown in Fig. 4C. TcMYH-overexpressor parasites showed a higher OD peak than wild type cells, and this higher amount of AP sites persisted longer in cells that overexpressed TcMYH than in control. This persistence of lesions in DNA was probably caused by a BER imbalance. AP sites data is in congruence with the DNA repair kinetics observed in QPCR technique (Quantitative Polymerase Chain Reaction; Fig. 6A and B). CL Brener epimastigotes had most nuclear lesions resolved between 2 and 10h of treatment (Fig. 6A), while we observed that at 6h AP sites in CL Brener's total DNA had already decreased to levels similar to non-treated cells (Fig. 4C). As in the quantification of AP sites, TcMYH-overexpressor strain also presented higher levels of DNA damage, as well as their persistence, when compared to WT (Fig. 6A and B). Additionally, MYH activity was reported as a cell death-signaling molecule in human cells (Markkanen et al., 2013). A model describes this function, in which DNA repair by MYH generates SSBs, leading to cell death by PARP or calpain signaling, making these SSBs cytotoxic specially on situations of oxidative burst. Indeed, this model explains why when there is oxidative burst the absence of MYH is beneficial for cell survival (Oka and Nakabeppu, 2011). Following this line of thinking, it has been suggested that hMYH has a role in mitochondrial dysfunction in Parkinson's disease (Fukae et al., 2007, Nakabeppu et al., 2007). However, it has not been established how MYH initiates apoptosis under some circumstances, while in other cases this protein protects cells from death.