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

Abstract

SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated. We investigated the role of endogenous SAMHD1 phosphorylation during the cell cycle. Thr-592 phosphorylation occurs first at the G1/S border and is removed during mitotic exit parallel with Thr-phosphorylations of most CDK1 targets. Differential sensitivity to the phosphatase inhibitor okadaic acid suggested different involvement of the PP1 and PP2 families dependent upon the time of the cell cycle. SAMHD1 turn-over indicates that Thr-592 phosphorylation does not cause rapid protein degradation. Furthermore, SAMHD1 influenced the size of the four dNTP pools independently of its phosphorylation. Our findings reveal that SAMHD1 is active during the entire cell cycle and performs an important regulatory role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication.

SAMHD1‐deficient fibroblasts from Aicardi‐Goutières Syndrome patients can escape senescence and accumulate mutations

“…6,7 In contrast to RNR, SAMHD1 is a nuclear protein. 10 Our findings agree with the recently reported participation of T592-phosphorylated SAMHD1 in protein/protein interactions at stalled replication forks, required for the ATR-CHK1 pathway and fork restart. 8 The protein accumulates in G0/G1 and quiescent cell states, which suggested that the enzyme may exert its activity mainly outside S phase.…”

“…In parental and SAMHD1-KO THP1 cells, the analysis of replication dynamics highlighted a pronounced reduction (P < .001) in fork rates, IOD, and cluster size in THP1-KO cells ( Figure 4A-D and Supplemental Table S2). 10 Finally, the proportion of paused/arrested forks was doubled in THP1-KO, although the difference with the parental cells lacked statistical significance (Supplemental Table S2). This feature may account for the ability of the SAMHD1-KO THP1 cells to proliferate at the same rate as the parental THP1 cells.…”

“…The individual lines behaved differently: P3 and P4 cells grew poorly similar to the AGS skin fibroblasts studied by Kretschmer and coworkers, 44 whereas P1 cells grew like the controls and P2 cells, although slow-growing, reached the stationary phase at a WT cell density (Figure 2A). We estimated the degree of senescence in AGS and WT cell cultures at early [10][11][12][13][14][15] and late [30][31][32][33][34][35][36][37][38][39][40] passages after isolation ( Figure 2B). Populations of P3 and P4 cells became senescent at early passages and contained 40% and 60% SA-β-Gal stained cells, respectively.…”

“…9 However, we recently showed that in S phase SAMHD1 is still functional and performs a significant regulatory role in the maintenance of dNTP pool balance during DNA replication. 10 Besides the effects on dNTP pool composition, in normal human fibroblasts siRNA-silencing of SAMHD1 inhibits cell proliferation and disturbs the G1/S transition. 11 The latter function of SAMHD1 permits the degradation of ssDNA stretches removed from the stalled forks preventing accumulation of ssDNA in the cytosol and induction of the interferon response.…”

“…Lack of the former function is considered to underlie several cancers by lowering the fidelity of DNA synthesis, lack of the latter to cause the congenital inflammatory Aicardi-Goutières Syndrome (AGS). 10 There is no in vivo evidence for anti-proliferative effects of SAMHD1 deficiency. Loss of SAMHD1 is accompanied by expansion of the dNTP pools, particularly large in non-dividing cells.…”