DNA polymerases are key proteins involved in genome replication and its maintenance. The different DNA repair pathways acting in the nucleus are well understood. However, little is known about how the mitochondrion repairs the damage to its genome and which DNA polymerases might localise to the organelle during genome repair. In Caenorhabditis elegans, DNA polymerase theta is considered a key enzyme in genome evolution and diversification, but little is known about the influence of this and other DNA polymerases in the nematodes aging. Moreover, whilst DNA polymerases are well conserved across eukaryotes, it is still not clear why some eukaryotes have lost some DNA polymerases during evolution when DNA damage affects all organisms. This thesis aims to investigate these questions by using a laboratory and in silico approach. It was observed that DNA polymerase lambda which is able to perform translesion DNA synthesis might have a mitochondrial localisation in mammalian cell lines under specific DNA damaging conditions, such as increased reactive oxygen species. This was observed by co-localisation studies which showed a positive co-localisation between DNA polymerase lambda and the mitochondrial protein TFAM. Although a mitochondrial target sequence was not predicted in DNA polymerase lambda protein sequence, it is possible that this DNA polymerase might locate to the mitochondrion using a non-canonical route.In C. elegans, DNA polymerase theta (polq-1) seems to have a more important role in the protection of the nematode genome than DNA polymerase eta (polh-1) by compensating in the absence of DNA polymerase eta. Moreover, polq-1 mutants seem to accumulate DNA mutations that are transmitted to next generation resulting in less viable eggs after exposure to specific DNA damaging agents, such as increased reactive oxygen species levels. It was observed that the central region of mammalian DNA polymerase theta might interact with other proteins during double-strand break repair in HEK293FT cells. However, the identification of those proteins by mass-spectrometry was not successful. Finally, DNA polymerase theta was found in eukaryotic groups which were thought to not have this DNA polymerase conserved in their genome, including chytrids (a fungal group). It is not clear why DNA polymerase theta was kept in the genome of these species, but it is possible that theta-mediated end-joining mediated by DNA polymerase theta contributes to their genome repair or it can be involved in genome evolution. This research revealed that specific translesion DNA polymerases, including Pol λ, might have a dual subcellular localisation in mammalian cells, however, further studies are needed to confirm these data. In addition, the data collected here revealed that Pol θ might be involved in the DNA repair or lower eukaryotes, such as chytrids. Evidence that additional translesion DNA polymerases might contribute to mitochondrial genome repair in mammals and in C. elegans was collected, but further studies are needed to conclude these findings. Overall, the translesion DNA polymerases studied here appeared to have an important and different roles across several eukaryotic phyla.