A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase.

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.

Extracting and Measuring dNTP Pools in Saccharomyces cerevisiae.

Regulation of dNTP pools in an intracellular environment is not only vital for DNA replication but also plays a major role in maintaining genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and altered regulation of RNR leads to imbalanced dNTP pools. Increased dNTP levels are mutagenic and have the potential to interfere with pathways that are involved in DNA replication, repair and DNA damage control. However, the mechanisms through which altered dNTP pools affect these pathways are poorly understood. Nonetheless, altered dNTP pools have been identified in a number of cellular contexts, including cancer. In order to interpret and analyze the effects of altered dNTP pools, we need quantitative information about dNTP pools in different genetic and environmental contexts in vivo. Here we describe a high-throughput fluorescence-based assay that uses a qPCR-based approach to quantify dNTP levels for use with Saccharomyces cerevisiae extracts.

Measurement of mitochondrial dNTP pools.

Because deoxyribonucleoside triphosphates (dNTPs) are the critical substrates for DNA replication and repair, dNTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique.

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.

Separation and identification of trinucleotide-melphalan adducts from enzymatically digested DNA using HPLC-ESI-MS.

Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery of the modified species than C(18). The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified cross-link formation in DNA, even though at low abundance and only a few adduct types were detected.

Self-cleavage of DNA in the presence of metal ions.

DNA is well known to be aggregated by metal ions including Mn ions, however, analysis of the aggregation process from a chemical aspect, which means identification of the product yielded during the process, has not been performed yet. On determination of what kinds of degraded materials were in the supernatant obtained on centrifugation of a DNA mixture aggregated under the conditions of 10 mM Mn ions ([Mn]/[P]=46.3) at 70 degrees C for 1 h, dAMP, dCMP, dGMP, and TMP produced through self-cleavage of DNA were found in the water-soluble part. These mononucleotides were purified by HPLC using TSKgel ODS-80Ts, and identified by LC-TOF/MS. The self-cleavage was effectively occurred under the conditions of more than 5 mM Mn ions, a reaction temperature of more than 70 degrees C, a reaction time of more than 30 min, and the use of DNA with a molecular weight of more than 140 bp. The self-cleavage was affected by the molecular size of the DNA.

Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection.

We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.

Detection and quantification of modified nucleotides in RNA using thin-layer chromatography.

Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50-150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and two-dimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.

Elimination of residual natural nucleotides from 3'-O-modified-dNTP syntheses by enzymatic mop-up.

Here, we describe a novel strategy called enzymatic "Mop-Up" that efficiently removes contaminating dNTPs from reverse-phase, high-performance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP syntheses. Enzymatic mop-up takes advantage of the high selectivity of DNA polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminating dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-nitrobenzyl)-dTTP syntheses, respectively. These data highlight the importance of natural nucleotide contamination when interpreting enzymatic incorporation data and provide an alternative hypothesis for the observed property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs addresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the base addition sequencing scheme (BASS).

Genetic information 'created' by archaebacterial DNA polymerase.

DNA polymerase catalyses replication of cellular DNA. The reaction requires a primer-template complex, and a new DNA chain grows from the 3' end of the primer along the template; no genetic information is created in this reaction. We demonstrate that DNA polymerase from Thermococcus litoralis, a hyperthermophilic marine Archaea, can synthesize up to 50000 bp of linear double-stranded DNA in the complete absence of a primer-template complex, indicating that genetic information is 'created.' The possibility of DNA contamination in the reaction mixture, which may serve as a primer and/or template, was vigorously excluded; for example, pretreatment of DNA polymerase with DNase I or extensive chromatographic purification of the substrate, deoxyribonucleoside 5'-triphosphates, did not abolish the primer-template-independent DNA synthesis. The DNA synthesized was (CTAGATAT)n, (TAGATATCTATC)n or a related sequence. Similar repetitive sequences are found in centromeric satellite DNA of many organisms. The significance of this ab initio DNA synthesis is that genetic information can flow from protein to DNA.

Regulation of T4 phage aerobic ribonucleotide reductase. Simultaneous assay of the four activities.

We have devised an assay procedure that permits simultaneous monitoring of the four activities of ribonucleotide reductase. Using this assay, we have compared the reduction of all four substrates by the T4 bacteriophage aerobic ribonucleotide reductase within different allosteric environments. Specifically, we compared the relative turnover rates by the enzyme when activated with "in vivo" concentrations of the known allosteric effectors versus activation by ATP alone. Consistent with the known allosteric properties of this enzyme, our results show that ATP does act as a general activator, although the rate of purine nucleotide reduction was approximately 5% of the rate for the pyrimidine nucleotides. However, addition of the allosteric effectors at their estimated physiological concentrations dramatically changed the relative rates of substrate reduction, creating a more "balanced" pool of products. Addition of the substrates at their respective in vivo concentrations further pushed rates of product formation toward a ratio similar to the base composition of the T4 genome. The similarity of the product profile produced under in vivo conditions to the genomic composition of T4 phage is discussed.

Application of spherical and other polymers in capillary zone electrophoresis: separation of antiviral drugs and deoxyribonucleoside phosphates by different principles.

Soluble polymers of linear chains with limited branching and spherical polymers (limit dextrins and sucrose, such as Dextran and Ficoll (Pharmacia Chemicals), yielding lower viscosities, are examined here for the separation of different nucleotides and several anti-AIDS drugs by capillary zone electrophoresis (CZE). The linear polymer forms a network but spherical polymers appear to create a second pseudo-phase. In general, they tend to enhance the solute mobility and reduce peak width; thus, they improve the column efficiency. We observe that the beads of a spherical polymer produce a pseudo-phase even in a very low polymer concentration. The proposed method involving a spherical polymer yields the best separation for twelve deoxyribonucleoside mono-, di- and triphosphates in ca. 10 min. Common anti-AIDS drugs (ddA, ddC, ddI, d4T, AZT) and an AZT metabolite (AZT-glucuronate) are resolved by using conventional micellar electrokinetic capillary chromatography (MEKC). These results not only offer fast and highly sensitive detection techniques for the pharmacokinetics of nucleotides, drugs, and their metabolites, but they also demonstrate an application of the proposed second pseudo-phase involving spherical polymer beads in CZE separations.

The trinucleotide repeat sequence d(GTC)15 adopts a hairpin conformation.

The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.

Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.

A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid. The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E. coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively.

beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei.

We have previously shown that the DNA of the unicellular eukaryote T. brucei contains about 0.1% of a novel modified base, called J. The presence of J correlates with a DNA modification associated with the silencing of telomeric expression sites for the variant surface antigens of trypanosomes. Here we show that J is 5-((beta-D-glucopyranosyloxy)-methyl)-uracil (shortened to beta-D-glucosyl-hydroxymethyluracil), a base not previously found in DNA. We discuss putative pathways for the introduction of this base modification at specific positions in the DNA and the possible contribution of this modification to repression of surface antigen gene expression.

Nucleotide misincorporation on DNA templates containing N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene.

An octadecadeoxynucleotide, modified site-specifically with N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene (dG-N2-AAF), was prepared by enzymatic synthesis from a comparably modified decamer and then used as a DNA template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I containing (exo+) or lacking (exo-) 3'-->5' exonuclease activity. Using exo- Klenow fragment and all four deoxynucleotide triphosphate (dNTPs), primer extension is blocked one base before and opposite dG-N2-AAF. A small fraction of the reaction product represents translesional synthesis, in which dAMP is incorporated opposite the lesion. Kinetic studies of base insertion and chain extension indicate that the frequency of dAMP insertion opposite dG-N2-AAF is higher than that of other deoxynucleotide monophosphates (dNMPs) and of N-(deoxyguanosin-8-yl)-2-(acetylamino)-fluorene (dG-C8-AAF); however, the rate of extension of dA.dG-N2-AAF from the 3' terminus was much lower than that of dA.dG-C8-AAF. We conclude that dG-N2-AAF is a miscoding lesion and capable of generating G-->T transversion mutations in cells.

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy.