A Mitochondrial Orthologue of the dNTP Triphosphohydrolase SAMHD1 Is Essential and Controls Pyrimidine Homeostasis in Trypanosoma brucei

The
maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis
through synthesis and degradation
is critical for accurate genomic and mitochondrial DNA replication
fidelity. Trypanosoma brucei makes
use of both the salvage and de novo pathways for
the provision of pyrimidine dNTPs. In this respect, the sterile α
motif and histidine–aspartate domain-containing protein 1 (SAMHD1)
appears to be the most relevant dNTPase controlling dNTP/deoxynucleoside
homeostasis in mammalian cells. Here, we have characterized the role
of a unique trypanosomal SAMHD1 orthologue denominated TbHD52. Our
results show that TbHD52 is a mitochondrial enzyme essential in bloodstream
forms of T. brucei. Knockout cells
are pyrimidine auxotrophs that exhibit strong defects in genomic integrity,
cell cycle progression, and nuclear DNA and kinetoplast segregation
in the absence of extracellular thymidine. The lack of TbHD52 can
be counteracted by the overexpression of human dCMP deaminase, an
enzyme that is directly involved in dUMP formation yet absent in trypanosomes.
Furthermore, the cellular dNTP quantification and metabolomic analysis
of TbHD52 null mutants revealed perturbations in
the nucleotide metabolism with a substantial accumulation of dCTP
and cytosine-derived metabolites while dTTP formation was significantly
reduced. We propose that this HD-domain-containing protein unique
to kinetoplastids plays an essential role in pyrimidine dNTP homeostasis
and contributes to the provision of deoxycytidine required for cellular
dTTP biosynthesis.