The NTP pyrophosphatase DCTPP1 contributes to the homoeostasis and cleansing of the dNTP pool in human cells

Abstract

The size and composition of dNTP (deoxyribonucleoside triphosphate) pools influence the accuracy of DNA synthesis and consequently the genetic stability of nuclear and mitochondrial genomes. In order to keep the dNTP pool in balance, the synthesis and degradation of DNA precursors must be precisely regulated. One such mechanism involves catabolic activities that convert deoxynucleoside triphosphates into their monophosphate form. Human cells possess an all-α NTP (nucleoside triphosphate) pyrophosphatase named DCTPP1 [dCTP pyrophosphatase 1; also known as XTP3-TPA (XTP3-transactivated protein A)]. In the present study, we provide an extensive characterization of this enzyme which is ubiquitously distributed in the nucleus, cytosol and mitochondria. Interestingly, we found that in addition to dCTP, methyl-dCTP and 5-halogenated nucleotides, DCTPP1 hydrolyses 5-formyl-dCTP very efficiently and with the lowest Km value described so far. Because the biological function of mammalian all-α NTP pyrophosphatases remains uncertain, we examined the role of DCTPP1 in the maintenance of pyrimidine nucleotide pools and cellular sensitivity to pyrimidine analogues. DCTPP1-deficient cells accumulate high levels of dCTP and are hypersensitive to exposure to the nucleoside analogues 5-iodo-2'-deoxycytidine and 5-methyl-2'-deoxycytidine. The results of the present study indicate that DCTPP1 has a central role in the balance of dCTP and the metabolism of deoxycytidine analogues, thus contributing to the preservation of genome integrity.

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

“…We have previously shown that media supplementation with an excess of deoxycytidine induces an increase in the expression of DCTPP1 in HeLa cells (12). Hence, we expected that exposure to decitabine might induce a similar modulation of enzyme levels in response to the accumulation of aza-dCTP.…”

“…Cell lines were cultured in a humidified atmosphere of 5 % CO 2 at 37 ºC. quantified and subjected to hydrolysis with formic acid as previously described (12).…”

“…To stop the reaction, samples were precipitated with methanol, centrifuged for 20 min at 16,000 g, dried and used for the quantification of the dNTP pool size as described (12).…”

“…This pool increased by two-fold after exposure to 100 µM of decitabine (1.9 pmol/10 6 cells). We hypothesize that the excess of dUTP might be generated as a result of the allosteric activation of dCMP deaminase by the abnormally high levels of dCTP that accumulate in these cells (12) rendering an excess of dUMP that is subsequently converted to dUTP.…”

“…DCTPP1 can also hydrolyze C5-modified dNTPs such as 5-halogenated, 5-methyl and 5-formyl deoxycytidines, and therefore may have an additional 'house-cleaning' function (12). The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also plays two roles, providing dUMP for de novo biosynthesis of dTTP and sanitizing the deoxynucleotide pool by the specific removal of dUTP (13)(14)(15).…”