Discussion

Among the three essential kDNA polymerases, POLIB and POLID have roles in replication, while the role of POLIC in kDNA maintenance remains unclear. POLIC RNAi resulted in loss of fitness (LOF) and a decrease in mini- and maxicircle copy number. RNAi also resulted in an ancillary kDNA phenotype, suggesting that POLIC may have roles besides its DNA polymerase activity. Additionally, POLIC forms foci at the antipodal sites (AP) during kDNA S-phase via unknown mechanisms. Here, we addressed the requirements of DNA polymerase activity and an uncharacterized N-terminal region (UCR) by complementing POLIC RNAi with overexpression of POLIC variants. Importantly, RNAi complementation with WT-POLIC rescued the LOF, restored kDNA copy number and AP foci formation. In contrast, complementation with an inactive-POLIC mutant caused a rapid LOF compared to the parental RNAi cell line, a loss of kDNA, and localization to the kDNA disk throughout the cell cycle, verifying that the polymerase domain is essential to maintain kDNA. Overexpression of inactive-POLIC in a WT background indicated a dominant-negative phenotype, while that with the UCR deletion did not impact fitness. Lastly, complementation with the UCR deletion could not rescue LOF and abolished AP foci formation, TdT incorporation, and caused a stark increase in ancillary kDNA compared to parental RNAi. Together these results show the UCR and DNA polymerase activity are both indispensable for maintaining kDNA integrity.