Mitochondrial DNA of trypanosomatid parasites is organized into a topologically complex structure, named kinetoplast kinetoplast DNA (kDNA). Replication, segregation and expression of kDNA involve an estimated ∼300 proteins, only a fraction of which have been identified and characterized. Here, we report the development of a genetic screen in Trypanosoma brucei to identify novel kDNA maintenance factors. Of the 20 highest-ranked genes identified, six are known kDNA maintenance factors. We selected one hit, Tb927.8.4240, a gene of previously unknown function, for further experimental characterization. Ultrastructure expansion microscopy using a tagged version of the protein reveals a dynamic localization during the cell cycle. RNA interference-mediated ablation of Tb927.8.4240 results in the progressive but incomplete loss of kDNA, with only a minor effect on the tripartite attachment complex, suggesting the protein is involved in kDNA replication but not segregation. The growth phenotype of Tb927.8.4240 ablation is fully rescued in a kDNA-independent genetic background, confirming a specific role in kDNA replication. In summary, we describe a functional genetic screen for the identification of kDNA maintenance factors in trypanosomes, validate one hit as a novel kDNA replication factor, and provide a prioritized hit list as a promising starting point for the future identification of additional factors.
Miskinyte et al. (Wed,) studied this question.