Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13.

The chain termination DNA sequencing procedure of Sanger et al. (1977) requires single-stranded DNA as template. M13 phage DNA exists as a single strand and therefore every DNA sequence cloned in M13 can be easily obtained in this form. Here we show that M13 single-stranded DNA pure enough to be used as a template for sequence determination can be prepared by simple centrifugation of the phage particle and extraction with phenol.     

Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-³H]-2′-deoxyuridine or [5-³H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-³H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.     

Light microscopic structure of DNA in solution studied by the 4′,6-diamidino-2-phenylindole staining method

Individual DNA molecules in solution can be visualized under a fluorescence microscope by using the DNA-binding dye 4′,6-diamidino-2-phenylindole and can be recorded on video as mobile structures (Morikawa & Yanagida, 1981). DNA in the presence of 10mm-MgCl₂ was found to adhere to the glass surface, so that 4′,6-diamidino-2-phenylindole-stained DNA can be filmed as still images. Fluorescence micrographs of DNA (bacteriophages T4, T3 and λ, yeast and chicken erythrocyte) taken by the present procedure are better in resolution than those obtained by video, showing structural details of DNA molecules hitherto not observed in solution. In the specimens prepared at the reduced shear stress, the folded particles and the short thick filaments were abundant. The shear stream extended them into the wavy and the straight thin filaments. The lengths of the thin filaments seen in viral DNA correlated well with those determined by electron microscopy. Our results suggest that a DNA molecule in solution forms a certain kind of supercoil of its own accord.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Simple and Rapid Enzymatic Method for the Synthesis of Single-Strand Oligonucleotides Containing Trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5′-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5′-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.     

Iododeoxyuridine administered to mice is de-iodinated and incorporated into DNA primarily as thymidylate.

Iododeoxyuridylic acid, a structural analog of thymidylic acid, is extensively de-iodinated in vivo by the enzyme thymidylate synthetase. Substantial amounts of the deoxyuridylic acid formed by this process are subsequently methylated and incorporated into DNA as thymidine. As a result, when mice are given tritiated iododeoxyuridine, most of the tritium incorporated into their DNA is present in thymidine rather than in iododeoxyuridine. Some, but not nearly as much, tritium from tritiated bromodeoxyuridine is also incorporated into DNA thymidine.     

Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.     

Synthesis and Properties of DNA Containing Cyclonucleosides

Here, we present efficient syntheses of the R and S diastereomers of 8,5′-cyclo-2′-deoxyadenosine and 6,5′-cyclo-2′-deoxyuridine. We incorporated these interesting nucleosides into DNA to study how the cyclo linkage affects the stability of duplex formation.     

4′-C-Aminomethyl-2′-deoxy-2′-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting the ability of a DNA/RNA duplex to activate RNase H

An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-β-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides.     

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase.

The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-³²P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, $\text{E. coli pol}$Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 2-Pyridone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O,4′-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin,and the effect of DNA replication inhibition

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-³H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.     

Versatile Solid Supports for Oligonucleotide Synthesis that Incorporate Urea Bridge

The universal solid support, USIII, representing a new and improved version of commercial USII, as well as 2 ′-deoxynucleoside and 2 ′-deoxy-2 ′-fluoronucleoside bound supports, incorporating a labile phenoxyacetyl fragment, was synthesized by an aminomethyl polystyrene carbamoylation with corresponding azides in the presence of aqueous triethylammonium bicarbonate. All three solid phases incorporate a stable urea tether, thus bridging the polymer and functional linker. These new matrices proved to be potent solid phases for the synthesis of DNA, RNA, or modified oligonucleotides as well as randomized mixed 2 ′-ribo/2 ′-deoxy-2 ′-fluoro-RNA libraries and/or DNA libraries, randomized with trinucleotides (codons).     

Recent developments in analytical methodology for 8-hydroxy-2'-deoxyguanosine and related compounds

When biomolecules such as proteins, lipids, and DNA are subjected to oxidative attack by free radicals or other reactive species, a number of measurable biomarkers may be produced. The study of oxidative DNA damage is valuable in research concerning cancer and aging. The current review includes methodology involving various separation science techniques for the analysis of DNA oxidation biomarkers, mainly 8-hydroxy-2'-deoxyguanosine. This review will present recent analytical developments with respect to sample preparation and instrumental considerations, noting key outcomes and biological relevance where appropriate.     

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.     

Methodological considerations and factors affecting 8-hydroxy-2′-deoxyguanosine analysis

Oxidative stress is related to a number of diseases due to the formation of reactive oxygen species (ROS). There are also several substances found in the occupational environment or as life style related situations that generates ROS. A stable biomarker for oxidative stress on DNA is 8-hydroxy-2′-deoxyguanosine (8-OH-dG).

A potential problem in the work-up and analysis of 8-OH-dG is oxidation of dG with false high levels as a result of analysis. This paper summarizes and discusses some of the critical moments in terms of auto-oxidation. The removal of transition metals, low temperatures, absence of isotopes (or 2′-deoxyguanosine) and incubation times are all important factors. Removal of oxygen is complicated while the problem is reduced if a nitroxide (TEMPO) is added during work-up. Certain reducing agents and enzymes could be critical if added during work-up.

The application of the ³²P-HPLC method to analyze 8-OH-dG is discussed. The ³²P-HPLC method is suitable for 8-OH-dG analysis and avoids several factors that oxidizes dG by removal of dG before addition of isotopes. Factors of crucial importance (columns, eluents, gradients and detection of ³²P) for the analysis of 8-OH-dG are commented upon and certain recommendations are made to make it possible to apply the ³²P-HPLC methodology for this type of analysis.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

Controlled oxidation of calf thymus DNA to produce standard samples for 8-Oxodeoxyguanosine analysis; Effects of freeze-drying,storage and hydrolysis conditions

Calf thymus DNA containing defined levels of 8-hydroxy-2′-deoxyguanosine (8-oxodG) was prepared by treatment with visible light in the presence of photosensitiser Ro 19-8022. The DNA was checked for stability; after freeze-drying, the amount of 8-oxodG did not increase during 6 weeks' storage at room temperature. However, freeze-drying itself can introduce additional oxidative damage. Two enzymic hydrolysis regimes (DNase I, phosphodiesterases I and II, and alkaline phosphatase; or P1 nuclease and alkaline phosphatase) give similar values for 8-oxodG.