Peter Reichard

Enzymatic Synthesis of Deoxyribonucleotides

The purification and properties of an enzyme system (“Fraction B”) isolated from Escherichia coli B were recently described (1,2). It was shown that Fraction B in the presence of adenosine triphosphate, Mg”, and reduced lipoate catalyzed the formation of deoxycytidine diphosphate from cytidine diphosphate and also the formation of deoxyuridine phosphate derivatives from uridine diphosphate (UDP). In addition, Fraction B was found to contain a specific pyrophosphatase that cleaved dUTP to dUMP and pyrophosphate. It was suggested that this latter enzyme might account for the absence of uracil in deoxyribonucleic acid. On the basis of these reactions, and others not previously reported in detail, it was postulated that the formation and transformations of deoxyuridine phosphates occurred according to the following scheme.

Role of effector binding in allosteric control of ribonucleoside diphosphate reductase

Abstract The allosteric effectors, ATP, dATP, dGTP and dTTP, regulate both the level of activity and the substrate specificity of ribonucleoside diphosphate reductase from Escherichia coli . The active enzyme consists of a complex of two non-identical subunits, proteins B1 and B2. Addition of negative effectors, such as dATP, leads to the formation of an inactive form which is probably a dimer of the active enzyme. We have now studied the binding of the effector nucleotides to the B1 and B2 subunits. Subunit B2 does not bind any of the nucleotides, while subunit B1, either alone or in combination with subunit B2, contains a total of four binding sites. These can be divided into two classes, each containing two sites. The first class (h-sites) has a high affinity for dATP and binds ATP, dATP, dGTP and dTTP; the second class (l-sites) has a lower affinity for dATP and binds only ATP and dATP. Binding of dATP to l-sites is considerably tighter in the presence of protein B2 than in the isolated protein B1. This suggests a mechanism for the generation of the inactive form of the enzyme through the binding of dATP. It appears that the l-sites are primarily involved in the regulation of the over-all activity of the enzyme, while binding of effectors to the h-sites influences the substrate-specificity . The binding of different nucleotides to h- and l-sites results in the formation of many distinct species of protein-effector complexes, each of which can be related to particular functional states of the enzyme. This suggests that the regulation of ribonucleoside diphosphate reductase requires the existence of many distinct conformational states.

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

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.

Ribonucleoside diphosphate reductase. Formation of active and inactive complexes of proteins B1 and B2.

Ribonucleoside diphosphate reductase from Escherichia coli B separates during purification into two catalytically inactive subunits, proteins B1 and B2. When the subunits are mixed, enzyme activity is readily regenerated. Sucrose gradient centrifugation indicated that protein B1 (sedimentation coefficient 7.8 s) and protein B2 (5.5 s) combined in the presence of magnesium ions to form a catalytically active complex. Under conditions which minimized dissociation this complex had a sedimentation coefficient of 9.7 s and consisted of equimolar amounts of the two subunits. The activity and substrate-specificity of the enzyme are modulated by nucleoside triphosphates which act as allosteric effectors. Stimulatory effectors, such as ATP and dTTP, did not appreciably change the sedimentation coefficient of the complex. However, in the presence of the negative effector dATP, at concentrations which inhibit enzyme activity, the 9.7 s complex was replaced by a heavy species with a sedimentation coefficient of 15.5 s. This heavy complex contained proteins B1 and B2 in equimolar amounts. A similar heavy complex was also formed with mixtures of other nucleoside triphosphates which inhibit the enzyme. On the other hand, the formation of the heavy complex was prevented by ATP at concentrations which reverse inhibition by dATP. We conclude from our results: (1) that the 9.7 s complex represents the active form of ribonucleoside diphosphate reductase; (2) that the 15.5 s complex represents an inactive form of the enzyme, and (3) that both complexes contain equimolar amounts of each subunit. From consideration of the differences in sedimentation coefficients we propose that the 15.5 s complex is a dimer of the 9.7 s complex.

Quantitation of cellular deoxynucleoside triphosphates

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.

The Ribonucleotide Reductase System of Lactococcus lactis CHARACTERIZATION OF AN NrdEF ENZYME AND A NEW ELECTRON TRANSPORT PROTEIN

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.

Origins of mitochondrial thymidine triphosphate: Dynamic relations to cytosolic pools

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.

Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage

Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.

p53R2-dependent Ribonucleotide Reduction Provides Deoxyribonucleotides in Quiescent Human Fibroblasts in the Absence of Induced DNA Damage

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-3H]cytidine, [6-3H]deoxycytidine, and [C3H3]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2–3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.

Crystal structure of a human mitochondrial deoxyribonucleotidase

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.