Pyrimidine deoxynucleotides

Regulation of the balance of the concentrations of the four deoxyribonucleotides depends on the properties of only two enzymes, the ribonucleotide reductase complex and deoxy-CMP deaminase. The balance between pyrimidine deoxynucleotides is brought about by the properties of the deoxy-CMP deaminase, which is inhibited by deoxy-TTP and stimulated by deoxy-CTP. The ribonucleotide reductase also possesses allosteric sites which bind all four deoxynucleotide triphosphates, the effect of which is to maintain approximately similar concentrations of all the triphosphates. 

This parasite also differs from T. foetus in that it lacks nucleotide reductase activity. Pyrimidine deoxynucleotides are obtained by salvage of deoxynucleosides by the deoxyribonucleoside phosphotransferase which acts not only on pyrimidine but also purine deoxyribonucleosides (22). 

It has been shown that purine-pyrimidine dinucleotides are considerably more add-labile than pyrimidine-pyrimidine dinudeotides and that the cytosine deox3uibose bond is more labile than the thymine deoxyribose bond. Thus, when thymidylic-uridylic add dinucleotide is hydrolyzed more thymidine 3, 5 -diphosphate is formed lhan uridine 3, 5 -diphos-phate [Eq. 56]. On the basis of these data an attempt was made to gain information on the distribution of pyrimidine deoxynucleotides in different preparations of DNA (255). Differences were detected in the rate of appearance of the pyrimidine deoxynucleoside dipho hates, i.e., early appearance from isolated pyrimidine deoxynucleotides and later appearance from adjoining pjuimidine deoxynucleotide sequences. This indicates that the distribution of pyrimidines in DNA is not random and differs from one preparation to another, even in samples of DNA with the same base composition. 

A sequence, in general, is the relative order of base pairs, whether in a fragment of a protein, DNA, a gene, a chromosome, or an entire genome. DNA is composed of two antiparallel strands of deoxynucleotides held together by hydrogen bonds between purine (adenine, A and guanine, G) and pyrimidine (thymidine, T uracil, U and cytosine, C) bases. 

Problems caused by an imbalance of the concentrations of deoxynncleotides In experiments with replicating cells in cnltnre, if the cnltnre medium contains an imbalance in the concentrations of the four deoxynucleotides, especially an imbalance between the pyrimidine deoxynncleotides, the mntation rate increases markedly. Dehcien-cies or low activities of at least three enzymes can resnlt in an imbalance of the nncleotides and do, in fact, give rise to disorders (Box 20.2). 

De novo synthesis of purines and pyrimidines yields the monophosphates IMP and UMP, respectively (see p. 188). All other nucleotides and deoxynucleotides are synthesized from these two precursors. An overview of the pathways involved is presented here further details are given on p. 417. Nucleotide synthesis by recycling of bases (the salvage pathway) is discussed on p. 186. 

We examine here the biosynthetic pathways of purine and pyrimidine nucleotides and their regulation, the formation of the deoxynucleotides, and the degradation of purines and pyrimidines to uric acid and urea. We end with a discussion of chemotherapeutic agents that affect nucleotide synthesis. 

It is worth noting that the fluorescence decays and quantum yields are the same for 2 -deoxynucleosides and 2 -deoxynucleotides in the case of purines (dA/dAMP and dG/dGMP) while for the pyrimidines (dC/dCMP and dT/TMP), the fluorescence quantum yields of nucleotides are higher and the fluorescence decays slower as compared to those of the corresponding nucleosides. This shows that the phosphate moiety does affect the excited state relaxation to a certain extent. 

The existence of metal intermediate complexes with deoxynucleotides has been elucidated by Eichhorn et al. (26). Proton NMR spectra of dAMP, dCMP, dGMP and dTMP show, especially for dAMP and dGMP, a strong reaction of Cu2+, although the interaction with the pyrimidines was markedly reduced. Further experiments employing 31P NMR spectroscopy show the broadening of the phosphate resonance of the deoxyribonucleotides of adenine and thymine (26). 

Table I. Second Order Rate Constants for Reactions of Cl2 with Pyrimidines, Purines and Deoxynucleotides at pH 2.7 in 10-1M Sodium Chloride Solution...

Without an accurate determination of the products of the electrolytic process, we cannot state definitively the structural features of a molecule required for electrodetection. However, we have expanded the work into the area of nucleotides, nucleosides and bases, and predicted that the bases would be inactive due to their lack of a ribose moiety, while either purine or pyrimidine nucleosides and nucleotides would be electro-active. It was observed that adenine and uracil (a purine and pyrimidine base respectively), were not detected by this method, while the nucleotides and nucloesides were responsive. Due to the inactivity of 2-deoxyribose we predicted that the 2 -deoxynucleotides would not respond, and this lack of response was observed with 2 -deoxyuridine. 

From Fig. 1 (a,b,c, and d) it is clear that good separation of each ribonucleotide and deoxynucleotide in all purine and pyrimidine series is achieved on a C g column, using isocratic elution with ammonium phosphate buffer (0.2M, pH 5.1). Fig. 2 (a,b,c, and d) illustrates the effect of periodate treatment on the ribo-nucleotide-deoxynucleotide mixture in each series. In the chromatography of ribonucleotides, the triphosphate always is eluted first, closely followed by the diphosphate, with the monophosphate being retained for a relatively longer period. A similar pattern emerges with the deoxynucleotides. After periodate treatment, we have noted a minor shift in the elution pattern. 

The separation of nucleotides and deoxynucleotides, previously a formidable task involving the fractional crystallization of heavy metal and alkaloid salts 102) has been made much easier by developments in analytical techniques. Ion-exchange methods may be used for the purification, isolation, and identification of both classes of nucleotides from hydrolysis mixtures 103), Countercurrent distribution 104) and starch 106) and cellulose-column 106) as well as paper-strip chromatography 107) have also proved to be useful in separating nucleotides from natural sources. Spectro-photometric procedures based on the characteristic ultraviolet absorption spectra of the purines and pyrimidines have been the most convenient method to locate, estimate, and identify the fractions obtained in the previous separations. Since the nucleotides are acid in nature, they are often named as acids, e.g., adenylic acid, cytidylic acid. The general constitution of the purine nucleotides (and by analogy the pyrimidine nucleotides) is demonstrated by their hydrolysis by acids to a purine and ribose (or 2-deoxyribose) monophosphate and by alkalies to the nucleosides and phosphoric acid. The order of the constituents in a purine nucleotide must, therefore, be ... 

There has been a further report (see Vol. 25, p. 265) on the use of nucleoside -3 -0-bis (l,l,l,3,3,3,-hexafluoro-2-propyl)phosphites as precursors for other analogues, including H-phosphonothioates and dinucleoside phosphorothioates,227 and the method outlined in Scheme 18 has been used to make di- and oligo-deoxynucleotide phosphorodithioates, with best results in the pyrimidine series.228 The (l p, / p) and (5p, Sp)- diastereomers of the bis-(phosphorothioate) of UpUpU have been prepared in a stereocontrolled manner.229... 

As an alternative mode of action, the anticancer activity of 6 may result from inhibition of a key enzyme in de novo pyrimidine biosynthesis, depleting nucleotide and presumably deoxynucleotide pools . Tumor death would result from inability to synthesize RNA and/or DNA. It is noteworthy that at least two of the three general classes of compounds known to inhibit this enzyme function by disruption of electron transport. Dup 785 reportedly is being studied to determine possible interference with electron transport . ... 

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