Discussion

The coordinated replication and segregation of the genome to daughter cells is an integral cellular process to ensure inheritance and the preservation of life. Several protein kinases (PKs) have been documented to regulate multiple steps in nuclear DNA replication in yeast and other eukaryotes. However, though information is emerging about the machinery and coordination of T. brucei nuclear replication, nothing is known about the putative PK regulation of the reaction. Identification of how nuclear replication is regulated would not only provide insight into the evolution of this essential process, but may open up new avenues for the therapeutic intervention of the diseases caused by T. brucei and other kinetoplasts. To address this, we pooled all bloodstream form (BSF) T. brucei cell lines that individually target every PK (183 in total) by inducible RNAi. The pool was then sorted, with and without RNAi induction, according to their cell cycle stage based on DNA content (G1, S-phase and G2/M) and relative read depth mapped (RITseq) over time and per cell cycle stage. This screen revealed PKs already known to be involved in cell cycle progression (e.g. transition from G1 to S: CRK1 and CRK2), as well as several novel PKs. Functional characterisation of two of the PK revealed that their kinase activity is presumably obsolete. Perhaps, rather a structural role is implicated in the replication of the nuclear DNA.