The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells

Abstract

Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as a component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots and is ubiquitous in human organs. Here we identify a general function of SAMHD1 in the regulation of dNTP pools in cultured human cells. The protein was nuclear and variably expressed during the cell cycle, maximally during quiescence and minimally during S-phase. Treatment of lung or skin fibroblasts with specific siRNAs resulted in the disappearence of SAMHD1 accompanied by loss of the cell-cycle regulation of dNTP pool sizes and dNTP imbalance. Cells accumulated in G1 phase with oversized pools and stopped growing. Following removal of the siRNA, the pools were normalized and cell growth restarted, but only after SAMHD1 had reappeared. In quiescent cultures SAMHD1 down-regulation leads to a marked expansion of dNTP pools. In all cases the largest effect was on dGTP, the preferred substrate of SAMHD1. Ribonucleotide reductase, responsible for the de novo synthesis of dNTPs, is a cytosolic enzyme maximally induced in S-phase cells. Thus, in mammalian cells the cell cycle regulation of the two main enzymes controlling dNTP pool sizes is adjusted to the requirements of DNA replication. Synthesis by the reductase peaks during S-phase, and catabolism by SAMHD1 is maximal during G1 phase when large dNTP pools would prevent cells from preparing for a new round of DNA replication. dNTP regulation | cell cycle arrest | dGTP pool

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…As expected, cyclin E was present at the G1/S border and decreased as cells advanced into S phase, cyclin A2 accumulated during S and into G2, while cyclin B increased in G2/mitosis. SAMHD1 was present in all the phases and actually increased in confluent G1- and serum-starved G0 cells (Figure 2(a)), consistent with previous results[10]. In G1 and G0 T592 was not phosphorylated.…”

“…Considering the low level of mRNA after 8 h from tetracycline removal, we calculated for the protein a half-life of at least 16 h. We assessed the phosphorylation of GFP-SAMHD1 using the antibody specific for pT592 and found that the phosphorylated form decreased similarly to the non-phosphorylated one (Figure 5(c)). In line with our previous data with SAMHD1-silenced fibroblasts where the protein declined slowly in the absence of SAMHD1 mRNA [10], these results suggest that in proliferating cells SAMHD1 is a stable protein and phosphorylation does not fast SAMHD1 turn-over.

10.1080/15384101.2018.1480216-F0005Figure 5.Turn-over of SAMDH1 in proliferating U2OS cells. A .

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“…Earlier, we suggested that T592 phosphorylation may be a signal for SAMHD1 degradation during S-phase [10,43]. Here we showed that SAMHD1 co-immunoprecipitates with Skp2 in a phosphorylation dependent manner but we could not detect an increase of SAMHD1 protein after inhibition of the proteasome or of protein neddylation.…”

“…In S-phase the induction of the R2 subunit of RNR and the phosphorylation of SAMHD1 at T592 by the S-phase CDK complexes suggested a concerted regulation between the two opposite enzymatic activities. In our previous work [10] we suggested that SAMHD1 exerts its activity mainly in G1-phase and in quiescent cells. Here we show that SAMHD1 regulates the DNA precursors during the entire cell cycle.…”

“…SAMHD1 is a component of the enzyme network that controls dNTP levels [10]. In mammalian cells the concentrations of dNTPs are regulated with cell division cycle progression.…”