Using expression of TcMYH fused to GFP it was

Using expression of TcMYH fused to GFP, it was observed that MYH is present in the nucleus and in the mitochondrion of T. cruzi (Fig. 5). It is known that MYH protein from human, rat and mouse exhibits both mitochondrial and nuclear localization (Ichinoe et al., 2004, Lee et al., 2004, Nishioka et al., 1999, Takao et al., 1999, Nakabeppu, 2001). This dual localization has been described only for mammalian cells. Therefore, TcMYH is the first non-mammalian MYH to be characterized as present in both cell compartments. TcMYH has predicted nuclear and mitochondria localization; however, we could not determine the precise sequence involved in the transport of the protein to these organelles. Future investigations will be necessary to determine how T. cruzi TcMYH could be differently directed to the nucleus and to the mitochondrion. MutY/MYH acts only after the replication process, since formation of the SRT1720 pair GO:A is only possible with DNA duplication (Hayashi et al., 2002, Oka and Nakabeppu, 2011). Boldogh et al. (2001) reported that the nuclear isoform of hMYH colocalized with BrdU (a thymidine analog used for the detection of proliferative cells) and with the nuclear antigen of cellular proliferation (PCNA). Like PCNA, hMYH levels increased about three to four times during S phase, compared to G1 phase, whereas levels of hOGG1 and the mitochondrial isoform of hMYH did not change during cell cycle. These studies suggest that MYH is directed to the replication fork to ensure that its activity takes place in the newly synthesized DNA strand. It was also described that the interaction with PCNA is critical for the DNA glycosylase activity of hMYH and Schizosaccharomyces pombe MYH (Parker et al., 2001, Hayashi et al., 2002, Chang and Lu, 2002). These data support the association between MYH function and DNA replication. The antipodal sites are points adjacent to T. cruzi kDNA, where the duplication of mini circles occurs right before cells enter S phase (Schamber-Reis et al., 2012). Thus, the presence of TcMYH in the antipodal sites occurs during S phase. Since the parasites used in the localization experiments were not synchronized in their cell cycle, some were marked in antipodal sites, while others were not (Fig. 5), which suggests that there is a control over TcMYH transport to the mitochondrion, which might occur during mitochondrial DNA replication. This control could be related to the fact that in T. cruzi cells, the AP endonuclease is located only in the nucleus (Sepulveda et al., 2014). As TcMYH is a monofunctional DNA glycosylase, an AP endonuclease would be necessary to fix AP sites generated by MutY activity. Once AP endonucleases are absent from the parasite's mitochondria, the repair of MutY-related AP sites could occur in a way similar to the repair of gaps generated by mitochondrial genome replication. In the last phase of mitochondrial replication, minicircles migrate to the antipodal sites, where the final reactions of minicircle duplication, including the repair of gaps by DNA polymerase β, occur (Liu et al., 2005). To further characterize TcMYH localization in T. cruzi, we performed QPCR assays to measure DNA lesions in nuclear and mitochondrial DNA of TcMYH-overexpressor and WT cells (Fig. 6A and B). Cells overexpressing TcMYH had higher levels of DNA lesions than the control in certain time points (Fig. 6A). The quantification of mitochondrial damage demonstrated a small increase in the level of DNA lesions in TcMYH-overexpressor strain (Fig. 6B), although the number of damage, and the difference between the strains are lower than those observed for the nucleus. This strain also had a lower survival, when compared to the wild-type strain, to MtOx reagent (Fig. 6C). These results suggest a role of TcMYH protein in mitochondrial DNA metabolism. Other BER proteins have already been described as acting in the mitochondrion of T. cruzi (Lopes Dde et al., 2008, Furtado et al., 2012) and the presence of TcMYH could indicate the presence of a specific repair system for the damages caused by the oxidative stress in this organelle. The results for nuclear and mitochondrial DNA damage support the survival curve (Fig. 4A), AP sites (Fig. 4C) and localization (Fig. 5) results, once they demonstrate that there is accumulation of DNA damage in TcMYH-overexpressor cells, in both organelles that contain DNA in a cell, and show that TcMYH is involved in nuclear and mitochondrial DNA metabolism.