Probing mechanism of metal catalyzed hydrolysis of Thymidylyl (3′-O, 5′-S) thymidine phosphodiester derivatives

Hydrolysis of nucleic acids is of fundamental importance in biological sciences. Kinetic and theoretical studies on different substrates wherein the phosphodiester bond combined with alkyl or aryl groups and sugar moiety have been the focus of attention in recent literature. The present work focuses on understanding the mechanism and energetics of alkali metal (Li, Na, and K) catalyzed hydrolysis of phosphodiester bond in modeled substrates including Thymidylyl (3′-O, 5′-S) thymidine phosphodiester (Tp-ST) (1), 3′-Thymidylyl (1-trifluoroethyl) phosphodiester (Tp-OCH₂CF₃) (2), 3′-Thymidylyl (o-cholorophenyl) phosphodiester (Tp-OPh(o-Cl)) (3) and 3′-Thymidylyl(p-nitrophenyl) phosphodiester (Tp-OPh(p-NO₂)) (4) employing density functional theory. Theoretical calculations reveal that the reaction follows a single-step (A_ND_N) mechanism where nucleophile attack and leaving group departure take place simultaneously. Activation barrier for potassium catalyzed Tp-ST hydrolysis (12.0 kcal mol^?1) has been nearly twice as large compared to that for hydrolysis incorporating lithium or sodium. Effect of solvent (water) on activation energies has further been analyzed by adding a water molecule to each metal ion of the substrate. It has been shown that activation barrier of phosphodiester hydrolysis correlates well with basicity of leaving group.

Evidence for the presence of ribonucleotides at the 5' termini of some DNA molecules isolated from Escherichia coli polAex2.

We have purified a set of small DNA molecules from various strains of exponentially growing Escherichia coli, including E. coli polAex2. This material included very short molecules (2 S), the nascent DNA (“Okazaki fragments”) and some longer molecules. Most of the [³H]thymidine incorporated during a brief period of labeling was found in the 5 S to 15 S Okazaki fragments. There was a large number of the 2 S molecules in the cell. The properties of the 5′ ends of these molecules were investigated using three procedures. (1) The DNA preparation, pulse-labeled with [³H]thymidine, was reacted with polynucleotide kinase and ATP to insure that all 5′ ends were phosphorylated. After subjection of the DNA to alkaline hydrolysis, the proportion of incorporated ³H pulse-label that became susceptible to digestion by spleen exonuclease was determined. In different experiments there was an increment of up to 20% in the amount of pulse-labeled E. coli polAex2 DNA that could be hydrolyzed by the exonuclease after treatment with alkali. (2) As in the preceding protocol, phosphorylation of the 5′ ends was assured by reaction with kinase and ATP; the preparation was then treated with alkali and the number of 5′-OH ends generated that could be labeled with ³²P using [γ-³²P]ATP and kinase in a second reaction was determined. The data indicated that 3 to 30% of the molecules could be labeled after alkali digestion, but not before. (3) The DNA molecules were reacted with kinase and [γ-³²P]ATP after having been exposed previously to alkaline phosphatase. The end-labeled molecules were then subjected to an alkaline hydrolysis and the resulting hydrolysate chromatographed on a polyethyleneimine-cellulose thinlayer plate. Alkali treatment was found to release 2′(3′),5′-ribonucleoside diphosphates from 1 to 30% of the molecules; pAp and pGp predominated. Control experiments showed that these ribonucleotides were covalently linked to the 5′ ends of polydeoxyribonucleotides. Curiously, the smaller the DNA molecule the less likely it was to possess a 5′-terminal ribonucleotide. Very few apparent RNA/DNA molecules were observed in the non-polAex2 strains tested. These observations are in part in agreement with previous reports, and we infer that at least some of the nascent E. coli polAex2 DNA molecules are initiated in vivo with a ribonucleotide primer. The relatively smaller proportion of molecules with apparent 5′-terminal ribonucleotides among the smaller DNA molecules and in strains other than E. coli polAex2 suggests to us that there may exist a mechanism for initiating DNA molecules that does not require an RNA primer.     

Facile Protection-Free One-Pot Chemical Synthesis of Nucleoside-5′-Tetraphosphates

A new, straightforward, reliable, and convenient protection-free one-pot method for the synthesis of 2′-deoxynucleoside-5′-tetraphosphate and ribonucleoside-5′-tetraphosphate is reported. The present synthetic strategy involves the monophosphorylation of a nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and subsequent hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide nucleoside-5′-tetraphosphate in moderate yield with high purity. A plausible mechanism is proposed to account for the formation of product.     

A kinetically important phosphoryl-enzyme intermediary in the intrinsic purine nucleoside-5'-diphosphokinase activity of Escherichia coli acetate kinase.

Initial rate studies of the intrinsic purine nucleoside-5′-diphosphokinase activity of Escherichia coli acetate kinase suggest that the kinetic reaction pathway is a ping-pong (or double-displacement) mechanism. Further evidence to support this mechanistic assignment was obtained through the use of the alternative substrate approach with ITP and GTP and by competitive inhibition studies with CrGTP and CrADP. That this diphosphokinase activity is intrinsic to the acetate kinase was demonstrated by the concomitant loss of the two activities when the phosphorylated form of acetate kinase was treated with 1 m hydroxylamine at pH 8. These data are fully consistent with the participation of an acyl-P intermediary in the acetate kinase and nucleoside diphosphokinase activities. The kinetic parameters suggest that the acetate kinase is a competent purine nucleoside-5′-diphosphokinase, but the metabolic significance of this function remains unassessed.     

Biosynthesis of polyribonucleotide phosphorylase and polyribonucleotides by E. Coli

The biochemical functions of Polyribonucleotide phosphorylase (PNP-ase; EC 2.7.7.8) has been studied The present work was aimed at studying the interrelation between PNP-ase biosynthesis and RNA content of E. coli cells under various conditions of bacterial growth, obtaining the biomass of E. coli with high PNP-ase activity as well as to study the possibility of secondary cultivation of E. coli cells for obtaining polyribonucleotides from nucleoside-5′-diphosphates The spcific PNP-ase activity increases at secondary cultivation, when the medium contains ribonucleoside-5′-diphosphates. Besides one may observe an extracellular polycondensation of nucleoside-5′-diphosphates It may be presumed that the PNP-ase is a multifunctional enzyme whose biological role may be confined to regulating the energetic processes connected with the metabolism of glucose, nucleosidephosphates and orthophosphate in the cells of E. coli.     

S1 nuclease of Aspergillus oryzae: a glycoprotein with an associated nucleotidase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae has been purified 1600-fold by a procedure designed to remove traces of contaminating phosphatases. The nearly homogeneous enzyme was found to be a glycoprotein with a carbohydrate content of 18%. At pH 4.5 the enzyme preparation hydrolyzed single-stranded DNA, RNA, 3′-AMP, and 2′-AMP at relative rates of 100, 52, 13, and 0.05, respectively. The 3′-nucleotidase activity of this single-strand specific nuclease is inhibited by single-stranded DNA but not by double-stranded DNA. Three forms of the enzyme, with isoelectric points of 3.35, 3.53, and 3.67, were observed on electrofocusing, and each form exhibited the same relative activity on single-stranded DNA and 3′-AMP. Enzymatic hydrolysis of nucleotides occurred over a broad range of pH, with maximal activity at pH 6–7. Ribonucleotides were hydrolyzed approximately 100-fold more rapidly than deoxyribonucleotides. A high degree of base specificity was not observed. The 3′-nucleotidase activity was stimulated by Zn²⁺, but not by other divalent cations tested.     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

Characterization of a ribonuclease from Anacystis nidulans infected with cyanophage as-1

In Anacystis nidulans the ribonuclease (RNase) activity is very low but is greatly increased upon phage-infection. A RNase was isolated and purified over 300-fold from A. nidulans cells infected by cyanophage AS-1. The enzyme did not attack single- or double-stranded DNA, was inactive on p-nitrophenyl phosphate or bis-p-nitrophenyl phosphate as substrates, and had neither 3′- nor 5′-nucleotidase activity. The approximate MW of the enzyme was 12000. Maximal enzyme activity was at pH 7.5. No absolute requirement for metal ions was observed, but Fe³⁺ stimulated and Co²⁺ and Ni²⁺ inhibited enzyme activity. The enzyme is an endonuclease which, upon exhaustive hydrolysis, produces mainly oligonucleotides (average chain-length: 3) with 3′-P termini. Analysis of the base composition of these oligonucleotides and determination of their 3′-terminal nucleosides, together with the investigation of the rate of hydrolysis of synthetic polyribonucleotides, have shown that the enzyme has a relative specificity for uridylic acid.     

Multiple forms of human dihydrofolate reductase messenger RNA: Cloning and Expression in Escherichia coli of their DNA coding sequence

The programming capacity for the synthesis of human dihydrofolic acid reductase in a rabbit reticulocyte lysate has been found to be greatly enhanced in the polysomal poly(A)-containing RNA from a methotrexate-resistant human cell variant (6A3), as compared to the RNA from its parental line (VA₂-B). A major fraction of this promoting activity is associated with a 3.8 × 10³ base RNA species detectable as a band in the ethidium bromide-stained electrophoretic pattern of the RNA from 6A3 cells, but not in the RNA from VA₂-B cells. Furthermore, sucrose gradient fractionation experiments have indicated that another substantial portion of the messenger activity is associated with RNA components around 10³ bases in size. Double-stranded complementary DNA synthesized from total poly(A)-containing RNA of 6A3 cells has been size fractionated, and both large (1400 to 3800 base-pairs) and small size complementary DNA (600 to 1400 base-pairs) species have been used separately to transform Escherichia coli χ2282 with pBR322 as a vector. Of 76 transformants obtained with the large size complementary DNA, identified by a differential colony hybridization assay, none has expressed the dihydrofolic acid reductase coding sequence in E. coli, as judged by resistance to trimethoprim. By contrast, eight trimethoprim-resistant transformants have been obtained using the small size complementary DNA, and their plasmids have been shown to contain the dihydrofolic acid reductase coding sequence by restriction mapping and DNA sequencing; moreover, immunoautoradiographic experiments have revealed the presence in the extracts of two of these transformants of a protein with the electrophoretic mobility and immunoreactivity of human dihydrofolic acid reductase. Restriction mapping and DNA transfer hybridization experiments have further indicated that the inserts of the chimaeric plasmids conferring trimethoprim resistance upon the host and of those lacking this capacity cover together a complementary DNA region of about 3.35 × 10³ base-pairs, in which the 564 base-pair dihydrofolic acid reductase coding stretch is located near the 5′ end of the sense strand. RNA transfer hybridization experiments using different cloned complementary DNA fragments as probes have shown the presence of three species of dihydrofolic acid reductase-specific messenger RNAs, with sizes of 3.8 × 10³, 1.0 × 10³ and 0.8 × 10³ bases, differing in the length of the 3′ untranslated region, in the poly(A)-containing RNA from two methotrexate-resistant variants, 6A3 and 10B3, and, in greatly reduced amounts, in the RNA from their respective parents, VA₂B and HeLa BU25.     

The mode of action of divalent cations on the RNase catalyzed hydrolysis of RNA and uridylyl (3'-5') uridine

Pseudo first order rate constants (k′) have been measured for the RNase A catalyzed hydrolysis of uridylyl (3′–5′) uridine at several ionic strengths and compositions. The k′ values are independent of Mg²⁺ concentration between 0 and 10 mM. This shows that for hydrolysis of RNA, in which Mg²⁺ concentration does change k′, the perturbation must be through binding of Mg²⁺ to the substrate RNA rather than to the enzyme RNase.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

An exoribonuclease from Saccharomyces cerevisiae: effect of modifications of 5' end groups on the hydrolysis of substrates to 5' mononucleotides.

Using poly(A) as a substrate, an exoribonuclease has been purified from the high-salt wash of ribosomes of $\text{Saccharomyces cerevisiae}$. The product of the reaction of the exoribonuclease is 5′ AMP. Hydrolysis of [³H](pA)₃[¹⁴C](pA)_n shows that both labels are released at the same rate, suggesting that the enzyme acts in a processive manner. Removal of the terminal phosphate of poly(A) with alkaline phosphatase reduces the rate of hydrolysis by 80%. Treatment of the terminally dephosphorylated poly(A) with polynucleotide kinase restores the activity. Two 5′ capped mRNA's have been tested and they are hydrolyzed slowly, if at all, by the enzyme. In contrast, phage T4 mRNA, ribosomal RNA, and encephalomyocarditis viral RNA are hydrolyzed at greater than 50% of the rate of poly(A).     

Zn2+ -Promoted Hydrolysis of 3′,5′-Dinucleoside Monophosphates and Polyribonucleotides. The Effect of Nearest Neighbours on the Cleavage of Phosphodiester Bonds

Abstract

Pseudo first-order rate constants for the Zn²⁺ -promoted cleavage of 15 different dinucleoside monophosphates, 4 different ribo homopogmers and RNA III from baker's yeast have been determined. Furthermore, the distribution of various nucleosides at the 3′-and 5′-terminus of the oligomeric hdrolysls products of RNA has been quantified. On these bases, the effect of nearest neighbours on the metal-ion-promoted hydrolysis of the internucleosidic phosphodiester bonds of RNA is discussed.     

Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells.

A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides.Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infected Escherichia coli cells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ-³²P]ATP. After selective degradation of the DNA portions, [5′-³²P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.     

Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2′-deoxyuridine 5′-monophosphate (pTFT). The conjugate allows for direct optical and ¹⁹F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.     

Amine-catalyzed elimination of beta-phosphoric esters from aldehydes derived from ribonucleic acid and model substrates.

The kinetics have been measured for several steps of the diamine-catalyzed elimination of the terminal nucleoside from periodate-oxidized RNA and from several model substrates. The general-acid-catalyzed, rate-determining step has a k_HA of 0.13 M^?1 min^?1 (HA = RNH₃⁺) for primary amines, and the specific-base-catalyzed reaction has a k_HH of 0.35 min^?1 (0.2 mm RNA) with ornithine catalysis and a k_HH of 0.077 min^? (0.2 mm RNA) with lysine catalysis. Lysine has a third catalysis component, with a k_AH of 12 min^?1 M^?2. The diamino acid α,γ-diaminobutyrate is not effective as a catalyst, due to cyclic gem diamine formation. Substituents on the 5′-phosphoric ester group do not affect the kinetics unless the substituent is a proton (e.g., as in AMP); thus, AMP is not an accurate model for this type of sequential degradation of RNA.There are two degradative pathways, the β-elimination path and a route that involves cleavage of the C-1′-0-C-4′ ether linkage before the phosphoric ester is eliminated. The direct β-elimination path predominates below pH 7.5, with a maximum near pH 6, and yields only one set of end products. Because of its rapid and predictable course, the latter reaction is preferred for sequential degradation of RNA. The structure of the catalytically active intermediate (general-acid-catalyzed reaction series) involves the primary amino group of ornithine (lysine) condensed with the dialdehyde terminus to form the carbinolamine, aldimine, and enamine intermediates leading to the elimination.The ether cleavage path is controlled by a specific-base (k_HB) intramolecular catalysis above pH 7, and a side reaction leads to lowered yields of phosphoric ester cleavage. A primary amine group is required, since 3-dimethylamino propylamine does not catalyze the ether cleavage.     

Erratum

RNA polynucleotide kinase has been shown to transfer [γ³²P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′-OH kinase products are polyadenylated. A sizeable fraction of the [γ³²P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [³²P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α³²P] UTP, (3) different nearest-neighbor distribution for RNA molecules > 12S and 12-3S, (4) relative insensitivity of the [γ³²P] incorporation to α-amanitin as compared with total RNA synthesis, (5) internal [³²P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [³²P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ³²P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′-OH RNA kinase in a ligation reaction.