Biosynthesis of 5-(4''5''-dihydroxypentyl) uracil as a nucleoside triphosphate in bacteriophage SP15-infected Bacillus subtilis.

The nucleoside triphosphate of 5-(4',5'-dihydroxypentyl)uracil (DHPU) was detected in the acid-soluble extract from bacteriophage SP15-infected Bacillus subtilis W23. No uracil was found in the DNA of either replicating or mature phage. Labeled thymidine added during phage DNA synthesis was incorporated into phage DNA. The presence of DHPU as a nucleoside triphosphate in the acid-soluble pool and the incorporation of thymidine into phage DNA suggest that both DHPU and thymine are incorporated into SP15 DNA via their nucleoside triphosphates. 5-Fluorodeoxyuridine inhibited biosynthesis of SP15 DNA, and this inhibition was reversed by thymidine, resulting in the synthesis of a DNA containing reduced amounts of fully modified DHPU. It is proposed that 5-fluorodeoxyuridine, or its metabolic product, inhibits a step in the biosynthetic pathway to the nucleoside triphosphate of DHPU.     

Synthesis of the Unusual DNA of Bacillus subtilis Bacteriophage SP-15

Cultures of Bacillus subtilis infected with phage SP-15 were examined to investigate the metabolic origin of two of the unique components of the phage DNA: the component responsible for the unusually high buoyant density in CsCl and the unusual pyrimidine, 5-(4', 5'-dihydroxypentyl) uracil (DHPU). Newly synthesized pulse-labeled DNA was light in buoyant density and shifted to the high density of mature phage DNA upon further incubation. Parental DNA was converted to a light-density intermediate form prior to replication. When labeled uracil, thymidine, or DHPU were added to infected cells, it was found that only uracil served as the precursor to DHPU and thymine in phage DNA. Analysis of the bases from hydrolyzed DNA of labeled phage or infected cells indicated that the uracil was incorporated into the DNA as such (presumably via deoxyuridine triphosphate) and later converted to DHPU and thymine at the macromolecular level. The sequence of events after phage infection appeared to be: (i) injection of parental DNA; (ii) conversion of parental DNA to a light form; (iii) DNA replication, yielding light DNA containing uracil; (iv) conversion of uracil to DHPU and thymine; and (v) addition of the heavy component.     

Effects of nucleic acid compounds on viability and cell composition of Bdellovibrio bacteriovorus during starvation

The effects of various exogenous nucleic acid compounds on the viability and cell composition of Bdellovibrio bacteriovorus starved in buffer were measured. In decreasing order of effectiveness, these compounds were found to decrease the rate of loss of viability and the loss of cell carbon, cell ribonculeic acid, and cell protein: glutamate > ribonucleoside monophosphates > ribonucleosides > deoxyribonucleoside monophosphates. Similar sparing effects were not observed with nucleic acid bases, deoxyribonucleosides, ribose, ribose-5-phosphate, deoxyribose, and deoxyribose-5-phosphate. Appreciable increases in the respiration rate over the endogenous rate did not occur when cell suspensions were incubated with individual or mixtures of nucleic acid compounds. Formation of ¹⁴CO₂ by cell suspensions incubated with carbon 14-labeled nucleic acid compounds indicated ribonucleosides and ribonucleoside monophosphates were respired and to a small extent, were incorporated into cell material of non-growing cells. The respired ¹⁴CO₂ was derived mainly from the ribose portion of these molecules. No respired ¹⁴CO₂ or incorporated carbon 14 was found with bdellovibrios incubated with other nucleic acid compounds tested, including free ribose. During growth of B. bacteriovorus on Escherichia coli in the presence of exogenous UL-¹⁴C-ribonucleoside monophosphates, 10–16% of the radioactivity was in the respired CO₂ and of the radioactivity incorporated into the bdellovibrios, only 40 to 50% resided in the cell nucleic acids. However, during growth on ¹⁴C-adenine,-uracil, or-thymidine labeled E. coli, only trace amounts of ¹⁴CO₂ were found and 90% or more of the incorporated radioactivity was in the bdellovibrio nucleic acids. It is concluded that bdellovibrio can use ribonucleoside monophosphates during growth and starvation as biosynthetic precursors for synthesis of both nucleic acids and other cell materials as well as catabolizing the ribose portion for energy purposes.Abbreviations HM buffer 5 mM N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (pH 7.6) containing 0.1 mM CaCl₂ and MgCl₂ - DNA deoxyribonucleic acid - RNA ribonucleic acid - Ar, Cr, Gr, Ur ribonucleosides of adenine, cytosine, guanine, uracil, respectively - dTr deoxythymidine - AMP, CMP, GMP, UMP ribonucleoside monophosphates of adenine, cytosine, guanine, and uracil, respectively - dTMP deoxythymidine monophosphate - ATP adenosine triphosphate - PFU plaque-forming units     

Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA

A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.     

The preparation of 5-cyanouracil and 5-cyano-2''-deoxyuridine from the corresponding 5-iodo derivative and cuprous cyanide.

5-Cyanouracil has been prepared in high yield from cuprous cyanide and 5-iodouracil. The deoxynucleoside has been similarly prepared form 5-iodo-2'-deoxyuridine and this has enabled these compounds to be labelled with (14-C) cyanide. Attempts have been made to incorporate 5-cyanouracil into Escherichia coli 15T and into Mycoplasma mycoides var. capri DNA under conditions in which several other 5-substituted uracils have been incorporated, but without success. Similarly 5-cyano-2'-deoxyuridine could not be incorporated into the DNA of T3 phage under conditions in which 5-bromo-2'-deoxyuridine is easily incorporated. These results suggest that the criteria for a 5-substituted uracil to be incorporated into DNA in vivo depends on some factor other than the size of the substituent.     

Facile Post-Synthetic Derivatization of Oligodeoxynucleotide Containing 5-Methoxycarbonylmethyl-2′-Deoxyuridine

Abstract

5-Methoxycarbonylmetyl-2′-deoxyuridine residue was incorporated into oligoDNAs containing either an exclusive thymidine residue (dT) or all four natural deoxynucleoside residues (dA, dG, dC, dT) via a phosphoramidite method. The treatment of the fully protected oligomer bound to controlled pore glass (CPG), with a variety of polyamine resulted in the release of the oligomer from CPG and the incorporation of the polyamine at the 5-position of the uracil component, simultaneously and in good yields.     

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.     

A novel, highly modified, bacteriophage DNA in which thymine is partly replaced by a phosphoglucuronate moiety covalently bound to 5-(4',5'-dihydroxypentyl)uracil

Bacteriophage SP-15, which infects Bacillus subtilis, contains a highly modified DNA in which 62% of its thymine residues are replaced by 5-(4',5'-dihydroxypentyl)uracil to which is attached a phosphoglucuronate via a phosphodiester linkage to one of the hydroxyl groups of the pentyl side chain. Glucose is also bound to this residue probably by glycosidic linkage to the other hydroxyl group of the pentyl side chain. In 0.3 M KOH at 37 degrees C, glucuronic acid 1-phosphate is slowly released from this DNA. After enzymatic or acid-induced dephosphorylation, this sugar was identified by chromatography in two thin layer chromatography systems, conversion to glucuronolactone under conditions known to lactonize glucuronic acid, and reaction in four colorimetric assays for hexuronic acids. Phage SP-15 DNA is the first DNA found to have a uronic acid moiety or a phosphate which is not part of the phosphodiester backbone. The glucuronic acid phosphate might be derived from uridine pyrophosphoglucuronic acid, whose glucuronic acid moiety is normally destined for synthesis of teichuronic acid in the host cell wall.     

A proton magnetic resonance investigation of the glycosyl torsion angle of uracil nucleosides and nucleotides.

The use of line-shape decomposition techniques permitted the small 5-bond (5-J51') and 4-bond (4-J61') proton-proton coupling constants of a series of uracil nucleosides and nucleotides to be determined accurately. From an analysis of these coupling constants we have determined that the uracil base is in a predominantly anti conformation in aqueous solution and the mean position is not substantially altered by phosphate substitution at the 2', 3', or 5' positions, by changing the furanose stereochemistry from a ribose to a deoxyribose or an arabinose, or by an increase in temperature of 43 degree C.     

Characterization of 3''----5'' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.     

Modified bases in tRNA: the structures of 5-carbamoylmethyl- and 5-carboxymethyl uridine.

The crystal structures of two nucleosides, 5-carbamoylmethyluridine (1) and 5-carboxymethyluridine (2), were determined from three-dimensional x-ray diffraction data, and refined to R = 0.036 and R = 0.047, respectively. Compound 1 is in the C3'-endo conformation with chi +5.2 degrees (anti), psiinfinity = +63.4 degrees and psialpha = +180.0 degrees (tt); 2 is in the C2'endo conformation with chi +49.4 degrees (anti), psiinfinity -60.5 degrees and psialpha +60.0 degrees (gg). For each derivative, the plane of the side chain substituent is skewed with respect to the plane of the nucleobase; for 1, the carboxamide group is on the same side of the uracil plane vis a vis the ribose ring; for 2, the carboxyl group is on the opposite side of this plane. No base pairing is observed for either structure. Incorporation of structure 1 into a 3'-stacked tRNA anticodon appears to place 08 within hydrogen bonding distance of the 02' hydroxyl of ribose 33, which may limit the ability of such a molecule of tRNA to "wobble".     

Configurational effects on conformational properties of cyclic nucleotides. I. Theoretical calculations of conformer preferences in α-nucleoside 3′,5′ cyclic monophosphates

A comparative study has been made of the configurational effects on the conformational properties of α- and β-anomers of purine and pyrimidine nucleoside 3′,5′,-cyclic monophosphates and their 2′-arabino epimers. Correlation between orientation of the base and the 2′-hydroxyl group have been studied theoretically using the PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The effect of change in ribose puckering on the base-hydroxyl interaction has also been studied. The result show that steric repulsions and stabilizing effects of intramolecular hydrogen bonding between the base and the 2′-hydroxyl (OH) group are of major importance in determining configurations of α-anomers and 2′-arabino-β-epimers. For example, hydrogen bonding between the 2′-hydroxyl group and polar centers on the base ring is clearly implicated as a determinant of syn-anti preferences of the purine (adenine) or pyrimidine (uracil) bases in α-nucleoside 3′,5′-cyclic monophosphates. Moreover, barrier heights for interconversion between conformers are sensitive to ribose pucker and 2′-OH orientations. The result clearly show that a change in ribose-ring pucker plays an essential role in relieving repulsive interaction between the base and the 2′-hydroxyl group. Thus a C2′-exo-C3′-endo (₂T³) pucker is favored for α-anomers in contrast with the C4′-exo-C3′-endo (₄T³) from found in β-compounds.     

Biosynthesis of thiamin. The precursor of the five-carbon unit of the thiazole moiety

The precursor of the thiazole moiety of thiamin in Candida utilis was identified. Radioactive C-2, 3, 4, 5 and 6 of glucose was incorporated into C-4', 4, 5, 5' and 5" of the thiazole. This experiment shows that the precursor of the five carbon unit of thiazole is a 5-carbon compound such as ribose or ribulose derived from glucose.     

Carbocyclic 5'-noruridine

A convenient preparation of (1'R,2'S,3'R,4'S)-1-(2',3',4'-trihydroxycyclopent-1'-yl)-1H-uracil (carbocyclic 5'-noruridine, 1) is described in 2 steps from the palladium complex of (+)-(1R,4S)-4-hydroxy-2-cyclopenten-1-yl acetate (3) and the sodium salt of uracil (2). Compound 1 was sought as a previously unknown member of the series of carbocyclic 5'-nor nucleosides needed as moieties for new oligomers. With 1 available, its antiviral properties and those of its enantiomer (5) are reported with 5 showing promising activity towards Epstein-Barr virus.     

Inhibitory potency of 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one nucleosides against uridine phosphorylase from mouse leukemic L1210 cells

The inhibitory activity of a series of novel sugar-modified nucleosides derived from 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one against uridine phosphorylase purified from mouse leukemic L-1210 cells was investigated. Significant activity was encountered with O2,2'-anhydro-5-benzylcytidine hydrochloride, 2',3'-dideoxy-5-benzyluridine, 2',3'-dideoxy-4-thiouridine and alpha- and beta-anomers of 5-benzyl-1-(2-deoxy-D-arabino-hexopyranosyl)uracil.     

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.     

Photooxidation of d(TpG) by riboflavin and methylene blue. Isolation and characterization of thymidylyl-(3',5')-2-amino-5-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-4H-imidazol-4-one and its primary decomposition product thymidylyl-(3',5')-2,2-diamino-4-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-5(2H)-oxazolone.

The major initial product of riboflavin- and methylene blue-mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen-saturated aqueous solution has previously been identified as 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino] 4H-imidazol-4-one (dlz). At room temperature in aqueous solution dlz decomposes quantitatively to 2,2-diamino-4-[(2-deoxy-beta-D-erythro- pentofuranosyl)amino]-5(2H)-oxazolone (dZ). The data presented here show that the same guanine photooxidation products are generated following riboflavin- and methylene blue-mediated photosensitization of thymidylyl-(3',5')-2'-deoxyguanosine [d(TpG)]. As observed for the monomers, the initial product, thymidylyl-(3',5')-2-amino-5-[(2-deoxy- beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one [d(Tplz)], decomposes in aqueous solution at room temperature to thymidylyl-(3',5')-2,2-diamino-4- [(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone [d(TpZ)]. Both modified dinucleoside monophosphates have been isolated by HPLC and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, chemical analyses and enzymatic digestions. Among the chemical and enzymatic properties of these modified dinucleoside monophosphates are: (i) d(Tplz) and d(TpZ) are alkali-labile; (ii) d(Tplz) reacts with methoxyamine, while d(TpZ) is unreactive; (iii) d(Tplz) is digested by snake venom phosphodiesterase, while d(TpZ) is unaffected; (iv) relative to d(TpG), d(TpZ) and d(Tplz) are slowly digested by spleen phosphodiesterase; (v) d(Tplz) and d(TpZ) can be 5'-phosphorylated by T4 polynucleotide kinase. The first observation suggests that dlz and dZ may be responsible for some of the strand breaks detected following hot piperidine treatment of DNA exposed to photosensitizers.     

UVB-Induced formation of intrastrand cross-link products of DNA in MCF-7 cells treated with 5-bromo-2'-deoxyuridine

Nucleoside 5-bromo-2'-deoxyuridine (BrdU), after being incorporated into cellular DNA, is well-known to sensitize cells to ionizing radiation and UV irradiation. We reported here, for the first time, the sequence-dependent formation of intrastrand cross-link products from the UVB irradiation of BrdU-treated MCF-7 human breast cancer cells. Our results showed that BrdU replaced more than 30% dT in genomic DNA after the cells were treated with 10 microM BrdU for 48 h. LC-MS/MS data revealed that more than 50% of the incorporated BrdU was consumed during UVB irradiation, of which more than half was dehalogenated to yield dU. Low-dose (5.0 kJ/m2) UVB irradiation of BrdU-treated cells yielded four intrastrand cross-link products, where the C5 of uracil is covalently bonded to the C8 of its neighboring 5' or 3' guanine to give G[8-5]U and U[5-8]G, respectively, and the C5 of uracil could couple with the C2 or C8 of its vicinal 5' adenine to give A[2-5]U and A[8-5]U, respectively. All the above cross-link products except A[2-5]U could also be induced in BrdU-treated cells upon UVB irradiation at a dose of 39 kJ/m2. We further demonstrated, by using LC-MS/MS, that the yield of G[8-5]U was much greater than the total yields of A[2-5]U and A[8-5]U. In addition, our results revealed that BrdU treatment stimulated considerably the UVB-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in vivo. The formation of these intrastrand cross-link products and 8-oxo-dG in vivo underscores the importance of these products in the photosensitizing effect of BrdU.     

On the fidelity of DNA replication. Effect of the next nucleotide on proofreading

The contribution of proofreading to the fidelity by which Escherichia coli DNA polymerase I copies natural DNA has been analyzed by two independent criteria. With phi X174 am 3 DNA as a template, there is approximately a 25-fold increase in noncomplementary base substitutions at position 587 when the concentration of the next correct nucleotide, dATP, is increased. Sequence analysis indicates that the mistakes represent misincorporation of C in place of T at position 587. This mutagenic response is presumed to result from a decrease in the probability of excision by the 3' leads to 5' exonuclease of Pol I and is considered within the context of current theories on proofreading. No enhanced mutagenicity is observed with avian myeloblastosis virus DNA polymerase, which lacks a 3' leads to 5' exonuclease. Using a second approach, an enhancement in mutagenesis as large as 30-fold is observed to result from the addition of deoxynucleoside monophosphates to the Pol I reaction. This mutagenicity occurs with any of the four deoxynucleoside monophosphates and is independent of a significant inhibition of DNA synthesis, thus supporting proofreading models in which sites of excision and incorporation are independent. The results of both approaches suggest that the exonucleolytic activity of Pol I can increase fidelity by approximately 30-fold on natural DNA, a value much higher than previous estimates with polynucleotide templates. The effect of the next correct nucleotide in decreasing accuracy provides an in vitro probe for screening eukaryotic cells for putative proofreading functions.