Pyrimidine requirements in deoxyuridine triphosphate nucleotidohydrolase deficient Trypanosoma brucei mutants

Trypanosomal all-alpha dUTPases are homodimeric enzymes that catalyze the hydrolysis of dUTP and dUDP to dUMP and PPi. Trypanosomes lack dCTP/dCMP deaminase and therefore strongly depend on dUDP/dUTP hydrolysis for dUMP production. Here we have addressed by gene replacement the consequences of elimination of dUTPase activity in bloodstream forms of Trypanosoma brucei. We first generated conditional DUT-knockout strains that allowed an effective decrease of dUTPase resulting in proliferation arrest, although gene repression could not be sustained long enough to cause lethality. Alternatively, DUT null mutants could be isolated in the presence of high levels of thymidine while exogenous supplementation with uracil, uridine or deoxyuridine could not complement metabolically the dUTPase deficiency. Upon thymidine removal, trypanosomes exhibited impaired proliferation and eventually died. These data establish a strict requirement for dUTPase in T. brucei viability and support a major role of the enzyme in the provision of pyrimidine nucleotides in kinetoplastids.

Trypanosomal dUTPase plays a dual biological role providing the unique precursor for the de novo dTTP biosynthesis and eliminating non-canonical dUTP from the nucleotide pool.83Highlights► Trypanosomal dUTPase catalyzes the hydrolysis of dUTP and dUDP to dUMP and PPi. ► dUTPase is essential for viability of Trypanosoma brucei bloodstream cells. ► Exogenous supply of thymidine can complement the dUTPase deficiency. ► Uracil, uridine or deoxyuridine do not revert the lethal phenotype of DUT mutants. ► dUTPase protects against antifolates by preventing dUTP pool expansion.