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
Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.
Nucleoside monophosphate phosphohydrolases or 5Ј-nucleotidases (members of EC 3.1.3.5 and EC 3.1.3.6) dephosphorylate non-cyclic nucleoside monophosphates to nucleosides and inorganic phosphate. Seven human 5Ј-nucleotidases with different subcellular localization have been cloned (Table I). Sequence comparisons show high homology only between cytosolic 5Ј-nucleotidase IA (cN-IA) 1 and B and between cytosolic 5Ј(3Ј)-deoxynucleotidase (cdN) and mitochondrial 5Ј(3Ј)-deoxynucleotidase (mdN). However, the existence of common motifs suggests a common catalytic mechanism for all intracellular 5Ј-nucleotidases. Some 5Ј-nucleotidases are ubiquitous (ecto-5Ј-nucleotidase (eN), cN-II, cdN, and mdN); others display tissue-specific expression (cN-I and cN-III).Here we summarize recent advances on the structure and cellular functions of the cloned 5Ј-nucleotidases. We also propose a revised nomenclature, agreed upon with other colleagues active in the nucleotidase field. Catalytic MechanismCrystal structures are known for human mdN (10) and cdN 2 and for Escherichia coli periplasmic 5Ј-nucleotidase (11), a homologue of eN. All intracellular nucleotidases share a DX-DX(V/T) motif critical for catalysis and show structural similarity to the haloacid dehalogenase superfamily of enzymes (10). eN belongs to a separate family that includes also 2Ј,3Ј-cyclic phosphodiesterases and apyrases (11).The crystal structure of mdN and work on the active site of cN-II form the basis for a reaction mechanism of intracellular 5Ј-nucleotidases (10, 12). The reaction creates a phosphoenzyme intermediate involving the first aspartate in the DX-DX(V/T) motif (12). A detailed scheme of the catalytic process derived from the crystal structure of mdN (10) involved both aspartates in the above motif (Fig. 1). The first (Asp-41) generates a pentacovalent phosphorus intermediate with similar basic organization as the intermediate detected in the structure of -phosphoglucomutase (13). The x-ray structure of cdN suggests a catalytic mechanism identical with that of mdN. Differences within the active sites account for differences in substrate specificity (10).2 Using the structurally important residues the best alignment was between the two deoxynucleotidases and cN-III (10). Two 5Ј-nucleotidases, cN-II and cN-III, exhibit phosphotransferase activity (for reviews see Refs. 14 and 15) possibly because of higher stability of the phosphoenzyme intermediate or faster exchange of the nucleoside product with the nucleoside acceptor. The active site of E. coli 5Ј-nucleotidase, the paradigm for eN, contains two zinc ions and the catalytic dyad . No phosphoenzyme intermediate is formed during catalysis, but a water molecule performs the nucleophilic attack on the phosphate (16). Properties, Detection, and Inhibition of 5-NucleotidasesAll 5Ј-nucleotidases have relatively broad substrate specificities. In agreement with the structural information on the active sites (10, 11), all family members except eN are absolutely dependent on magnesium for activity. Table II s...
5' nucleotidases are ubiquitous enzymes that dephosphorylate nucleoside monophosphates and participate in the regulation of nucleotide pools. The mitochondrial 5'-(3') deoxyribonucleotidase (dNT-2) specifically dephosphorylates dUMP and dTMP, thereby protecting mitochondrial DNA replication from excess dTTP. We have solved the structure of dNT-2, the first of a mammalian 5' nucleotidase. The structure reveals a relationship to the HAD family, members of which use an aspartyl nucleophile as their common catalytic strategy, with a phosphoserine phosphatase as the most similar neighbor. A structure-based sequence alignment of dNT-2 with other 5' nucleotidases also suggests a common origin for these enzymes. Here we study the structures of dNT-2 in complex with bound phosphate and beryllium trifluoride plus thymidine as model for a phosphoenzyme-product complex. Based on these structures, determinants for substrate specificity recognition and the catalytic action of dNT-2 are outlined.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.