Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Plants salvage deoxyribonucleosides in mitochondria.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides into the corresponding 5'-monophosphate deoxyribonucleosides to supply the cell with nucleic acid precursors. In mitochondrial fractions of the model plant Arabidopsis thaliana, we detected deoxyadenosine and thymidine kinase activities, while the cytosol fraction contained six-fold lower activity and chloroplasts contained no measurable activities. In addition, a mitochondrial fraction isolated from the potato Solanum tuberosum contained thymidine kinase and deoxyadenosine kinase activities. We conclude that an active salvage of deoxyribonucleosides in plants takes place in their mitochondria. In general, the observed localization of the plant dNK activities in the mitochondrion suggests that plants have a different organization of the deoxyribonucleoside salvage compared to mammals.

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa.

BACKGROUND AND AIMS: Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. METHODS: Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H(2)O(2) production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). KEY RESULTS: Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H(2)O(2), γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. CONCLUSIONS: The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers.

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [(14)C]formate, [2-(14)C]glycine and [2-(14)C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP --> IMP --> inosine --> hypoxanthine --> xanthine and GMP --> guanosine --> xanthosine --> xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.

Anaerobic growth of Bacillus mojavensis and Bacillus subtilis requires deoxyribonucleosides or DNA.

Bacillus mojavensis strains JF-2 (ATCC 39307), ROB2, and ABO21191(T) and Bacillus subtilis strains 168 (ATCC 23857) and ATCC 12332 required four deoxyribonucleosides or DNA for growth under strict anaerobic conditions. Bacillus licheniformis strains L89-11 and L87-11, Bacillus sonorensis strain TG8-8, and Bacillus cereus (ATCC 14579) did not require DNA for anaerobic growth. The requirement for the deoxyribonucleosides or DNA did not occur under aerobic growth conditions. The addition of a mixture of five nucleic acid bases, four ribonucleotides, or four ribonucleosides to the basal medium did not replace the requirement of B. mojavensis JF-2 for the four deoxyribonucleosides. However, the addition of salmon sperm DNA, herring sperm DNA, Escherichia coli DNA, or synthetic DNA (single or double stranded) to the basal medium supported anaerobic growth. The addition of four deoxyribonucleosides to the basal medium allowed aerobic growth of B. mojavensis JF-2 in the presence of hydroxyurea. B. mojavensis did not grow in DNA-supplemented basal medium that lacked sucrose as the energy source. These data provide strong evidence that externally supplied deoxyribonucleosides can be used to maintain a balanced deoxyribonucleotide pool for DNA synthesis and suggest that ribonucleotide reductases may not be essential to the bacterial cell cycle nor are they necessarily part of a minimal bacterial genome.

Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers.

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced tumors and DNA adduct formation in the mammary glands of female Sprague-Dawley rats by the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate.

We synthesized a novel organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (XSC), possessing low toxicity by comparison with inorganic Na2SeO3, and several other synthetic organoselenium compounds (K. El-Bayoumy, Cancer Res., 45: 3631-3636, 1985). We tested the effect of XSC treatment during the initiation phase on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma formation. A semipurified high-fat diet containing 80 ppm of XSC (40 ppm as selenium) was fed to 6-wk-old virgin female Sprague-Dawley rats for 2 wk, starting 1 wk before and ending 1 wk after carcinogen treatment. At 7 wk of age, rats were given a single dose of DMBA (5 mg) in 0.2 ml of olive oil by gastric intubation; the experiment was terminated 16 wk later. The development of mammary tumors in those rats that received XSC-supplemented diets was significantly inhibited when compared with the control group (fed the same diet without XSC supplements). This was evident from tumor incidence (percentage of tumor-bearing rats, 88 versus 20) and multiplicity of tumors (mean number of tumors/rats, 3.96 versus 0.28). The finding that XSC acts as a chemopreventive agent in the DMBA mammary tumor model prompted us to examine the effect of dietary XSC on DMBA-DNA binding in both the liver and mammary tissue under conditions identical to those described above for the bioassay. Rats (four/group) were killed 6, 24, 48, and 168 h after [3H]DMBA (5 mg/rat; specific activity, 51.2 mCi/mM) administration. Liver and mammary tissue were obtained and DNA was isolated. Dietary XSC was found to inhibit total DMBA-DNA binding in the mammary tissue, but not in the liver. The most profound effect was observed at early time points, i.e., 24 to 48 h after [3H]DMBA administration. The inhibition in total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol-epoxides of DMBA; these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine adducts on the basis of their chromatographic characteristics on high-pressure liquid chromatography and on a boronate affinity column. The inhibition of the DMBA-DNA binding in the target tissue provides a plausible explanation for the chemopreventive effect of XSC during the initiation stage of carcinogenesis.

Substrate binding domain of murine leukemia virus reverse transcriptase. Identification of lysine 103 and lysine 421 as binding site residues.

The substrate deoxynucleoside triphosphate (dNTP) binding site of Moloney murine leukemia virus (M-MuLV) reverse transcriptase was labeled with pyridoxal 5'-phosphate (PLP), a substrate binding site-directed reagent for DNA polymerases (Modak, M. J. (1976) Biochemistry 15, 3620-3626). Treatment of M-MuLV reverse transcriptase with PLP results in the loss of RNA-dependent DNA polymerase activity, but has no effect on ribonuclease H activity. Neither template-primer nor substrate dNTP alone shows any protective effect from PLP-mediated inactivation. However, the presence of both template-primer and complementary substrate dNTP significantly protects M-MuLV reverse transcriptase from PLP inhibition. Using tritiated sodium borohydride to label the pyridoxylated enzyme, approximately 4 mol of PLP were incorporated per mol of enzyme. In the presence of template-primer and the complementary dNTP, however, only 2 mol of PLP were incorporated. Comparative tryptic peptide mapping of enzyme, modified in the presence and absence of substrates by PLP reaction on C-18 reverse phase columns, indicated the protection of two peptides from pyridoxylation in the presence of substrate triphosphate. These two peptides were further purified and characterized by amino acid analyses and sequencing and were found to span residues 103 to 110 and 412 to 425 in the primary amino acid sequence of M-MuLV reverse transcriptase. Furthermore, Lys-103 of peptide I and Lys-421 of peptide II were found to be the targets of pyridoxylation, indicating that these 2 lysine residues are involved in substrate dNTP binding in M-MuLV reverse transcriptase.

Phosphorylation of 5-bromodeoxycytidine in cells infected with herpes simplex virus.

5-Bromodeoxycytidine is phosphorylated to 5-bromodeoxycytidine 5'-monophosphate in extracts of cells infected with herpes simplex virus but not in extracts of uninfected cells. The conversion of 5-bromodeoxycytidine to nucleotides and its utilization for DNA synthesis in uninfected cells occurs by deamination of 5-bromodeoxycytidine to 5-bromodeoxyuridine followed by phosphorylation of 5-bromodeoxyuridine to 5-bromodeoxyuridine 5'-monophosphate. In contrast, in cells infected with herpes simplex virus, 5-bromodeoxycytidine is phosphorylated directly to 5-bromodeoxycytidine 5'-monophosphate, which can then be deaminated to 5-bromodeoxyuridine 5'-monophosphate and incorporated into DNA. These results indicate a difference in the substrate specificity of nucleoside kinases induced by herpes simplex virus and the enzymes present in uninfected cells. It is suggested that this difference in substrate specificity between virus-induced and host-cell enzymes may allow selective chemotherapy of herpes simplex infections with 5-bromo- or 5-iododeoxycytidine.