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

## TL;DR

A DNA polymerase from the fungus Aspergillus fumigatus shows template-independent DNA synthesis, similar to vertebrate enzymes, suggesting this ability evolved earlier than previously thought.

## Contribution

The discovery of a fungal terminal deoxynucleotidyl transferase (TdT) challenges the belief that template independence evolved only in metazoans.

## Key findings

- AfPolX1 from Aspergillus fumigatus exhibits terminal transferase activity under physiological conditions.
- AfPolX1 can incorporate ribonucleotides and modified nucleotides, similar to vertebrate TdT.
- The findings suggest template-independent DNA synthesis originated in early eukaryotes.

## Abstract

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.

Graphical Abstract