Aspergillus fumigatus PolX1 is an early ancestor of vertebrate terminal deoxynucleotidyl transferases

X-family DNA polymerases (PolXs) perform essential roles in repair and maintenance of the genome. One branch of the PolXs have evolved to function as terminal transferases, extending DNA ends in a template-independent manner, unusual for polymerases. To date, template independence has been shown exclusively in metazoans. We analysed PolXs to determine the phylogenetic evolution of the terminal transferase function in fungal PolXs. We have identified and characterised a PolX from the saprophytic fungus Aspergillus fumigatus, named AfPolX1, that demonstrates inherent terminal transferase ability under physiologically relevant conditions. This is the first report for a fungal terminal deoxynucleotidyl transferase (TdT). Our findings indicate that template-independent ‘creative’ synthesis evolved earlier than previously thought and can be traced as far back as the early Polµ’s of multicellular fungi. We further show that like TdT, AfPolX1 is capable of introducing ribonucleotides and various nucleotides with 2′ ribose modifications, giving credence to the idea that the structural features necessary for PolXs observed promiscuous behaviour during template independence existed in the PolXs of early eukaryotes. Our findings suggest AfPolX1 as a promising candidate for use in enzymatic oligonucleotide synthesis.