Abstract
To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5′-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity.
Highlights
The accuracy of DNA replication is essential for the maintenance of genome integrity
We sought to unveil in different cell types the role of the enzyme in the homeostasis of canonical nucleotide pools and cell viability and how it interrelates to other enzymes involved in pyrimidine metabolism
Cell cycle analysis of dCTP pyrophosphatase 1 (DCTPP1)-knockdown MCF-7 cells revealed an increase in the percentage of cells in the G1 phase and a decrease in the proportion of cells in S and G2/M, indicating a delay in the progression of G1 to S, the latter being the active phase for deoxyribonucleoside triphosphates (dNTPs) biosynthesis
Summary
The accuracy of DNA replication is essential for the maintenance of genome integrity. Enzymatic hydrolysis of damaged nucleotides cleanses the DNA precursor pool by eliminating potentially mutagenic modified nucleotides. Such is the case of the human enzyme MTH1 responsible for the elimination of 8-oxo-dGTP [9]. In this respect, the recently characterized human all-α NTP pyrophosphatase DCTPP1 (dCTP pyrophosphatase 1) catalyzes the hydrolysis of dCTP into dCMP and pyrophosphate, a conversion which has been proposed to keep the cellular dCTP pool balanced [10]. We have previously reported that DCTPP1-deficient cells accumulate high levels of dCTP and are hypersensitive to exposure to the
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