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.

Keywords: cell cycle arrest; dGTP pool; dNTP regulation.

Fig. 1.

Expression of SAMHD1 in cultured human cells. (A) Relative levels of SAMHD1 mRNA and protein in different cell lines. The relative amount of mRNA was measured by RT real-time PCR in transformed cell lines (THP1, HEK293, and Jurkat) and in nontransformed lung and skin fibroblasts. SAMHD1 protein was detected by immunoblotting in extracts from transformed cells, lung and wild-type skin fibroblasts, and skin fibroblasts mutated for p53R2 or the mitochondrial thymidine (TK2) or deoxyguanosine (dGK) kinases. (B) Immunoblots of SAMHD1 from proliferating (P), confluent (C), and quiescent (Q) WT skin and lung fibroblasts. Asterisk marks an unspecific band (loading control). (C) Immunofluorescence shows nuclear localization of SAMHD1 (green) and cytosolic localization of the R1 (green) and R2 (red) subunits of RNR in lung fibroblasts. (D) Inverse relation between frequency of S-phase cells (filled circle) and abundance of SAMHD1 protein (open square) in proliferating cultures of lung fibroblasts. (E) Content of SAMHD1 in quiescent lung (black) and skin (gray) fibroblasts relative to the amount at confluency. The immunoblot shows the increasing SAMHD1 signal in lung fibroblasts.

Fig. 2.

Decline and recovery of SAMHD1 mRNA and protein during siRNA transfection and subsequent removal of siRNA. Proliferating cultures of lung fibroblasts were transfected for 48 h with anti-SAMHD1 siRNA 24 h after seeding. Cells were then replated in siRNA-free medium and grown for 14 d with medium changes every 3–4 d. SAMHD1 mRNA (open circle) and protein (filled diamond) were measured at the indicated times. Bars indicate range of values in two similar experiments.

Fig. 3.

Effect of SAMHD1 silencing on the growth of lung fibroblasts. (A) Cell growth in cultures transfected for 24–72 h with anti-SAMHD1 (open circle) or control (filled circle) siRNAs. Data from three experiments. Bars are SEs. (B) Percent of G1 (black), S (gray), and G2/M (white) cells in transfected cultures. The frequency of S-phase cells is indicated above each control (C) and silenced (Si) sample. (C) Frequency of BrdU-positive cells in cultures transfected with control (filled circle) or anti-SAMHD1 (open circle) siRNAs for 18–48 h and incubated with BrdU during the last 30 min. Data are from two identical experiments. At least 500 cells were scored for each time point. The frequencies were compared by 2 × 2 χ² analysis. *P < 0.05; **P < 0.001. Bars are SEs.

Fig. 4.

Effect of SAMHD1 silencing on dNTP pools of cycling lung fibroblasts. Relation between dNTP pool sizes and loss of S-phase cells in cycling cultures during transfection with two separate anti-SAMHD1 siRNAs (open square, open triangle) or with control siRNA (closed circle).

Fig. 5.

Ratios between pool sizes of SAMHD1-silenced and control lung fibroblasts. (A) Proliferating cultures transfected for 24 (white), 48 (gray), or 72 (black) h with control or anti-SAMHD1 siRNAs. Data are from three independent experiments. (B) Quiescent cultures of lung (broad stripes), WT (gray), or p53R2-mutated (thin stripes) skin fibroblasts transfected for 10 d with siRNAs during serum starvation. Data are from four to six experiments per cell line. All bars are SEMs.

Fig. 6.

Slow recovery of growth and cell cycle progression in lung fibroblasts after SAMHD1-silencing. (A) Cell growth of control (filled circle) and SAMHD1-silenced (open circle) lung fibroblasts replated in siRNA-free medium after 48 h transfections. (B) Frequencies of G1- (squares) and S-phase (circles) cells in control (filled square, filled circle) and silenced (open square, open circle) cultures. Bars in A and B show range of values at identical time points in two independent experiments. (C) Abundance of SAMHD1 (x), R2 (filled square), and p53R2 (filled triangle) at the indicated days of culture without siRNA. Left reports in aribitrary units the quantification of the immunoblots on the right. Notice the parallelism between R2 expression and frequency of S-phase cells (open circle) in the cultures.

Fig. 7.

Persistence of large dNTP pools in lung and skin fibroblasts after siRNA silencing of SAMHD1. Mean ratios between sizes of dGTP (filled circle), dCTP (open circle), dTTP (filled square), and dATP (open triangle) pools in silenced and control cells during 14 d without siRNA. Data from experiments with lung fibroblasts (Fig. 6 and Fig. S4A) and skin fibroblasts (Fig. S3 and Fig. S4B). Bars show range of values.

Fig. 8.

The pools of the dNTPs required for DNA replication are regulated by synthetic and catabolic enzymes. Synthesis occurs by (i) de novo synthesis by RNR in the cytosol and (ii) salvage of deoxynucleosides by deoxynucleoside kinases in the cytosol and mitochondria. A stepwise catabolism of dNTPs to deoxynucleosides occurs with the final intervention of 5′-deoxynucleotidases. Phosphorylases and deaminases further degrade deoxynucleosides in the cytosol. The triphosphohydrolase SAMHD1 is located in the nucleus and degrades dNTPs directly to deoxynucleosides.