Adenine incorporation into nucleic acids

Several analogues of adenine or adenosine are reported to be incorporated into nucleic acids 2-fluoroadenosine [342], tubercidin [190, 192, 342a], toyacamycin [193,342a], sangivatnycin [342a, b], cordycepin [168,343,344], 4-aminopyrazolo[3, 4-d] pyrimidine [119], formycin [344a], and 9- -D-arabinofuranosyladenine [152, 154], The evidence for the incorporation of 9-(3-D-arabinofuranosyladenine has been questioned [345]. 

B-4) Mercaptopurine is an antitumor drug that looks like the purine adenine and acts partly by interfering with its incorporation into nucleic acids. 

Adenosine was shown to be incorporated into nucleic acid purines, but to only about half the extent of adenine itself (L2). The incorporation of guanosine was also studied and here again the guanine derivative was a poorer precursor than the adenine derivative, but the utilization of guanosine was considerably greater than that of the aglycone (L22). The administration of the adenine nucleotides revealed that these compounds, whether as the 3 isomer or the 5 isomer, were poorer precursors than the free base adenine (Wl). With all nucleotides studied, the incorporation of the 5 isomer was less extensive than of the 3 isomer (R9). Roll et al. have shown that guanine nucleotides are considerably... 

From the species names, one may recognize some of the various participants in purine metabolism. The important purines guanine (Gua) and adenine (Ade) are both synthesized from inosine monophosphate (IMP). They may then be derived to (d)GTP and (d)ATP, respectively, prior to incorporation into nucleic acids like DNA and RNA. This simple explanation betrays the true complexity of purine metabolism, and elucidating the properties of such complex systems depends on sophisticated computational tools, which we describe presently. 

The cytokinins themselves cannot be incorporated into nucleic acids, but adenine residues in tRNA can be converted to cytokinin moieties. Roughly 0.05 to 0.1% of tRNA bases are estimated to be cytokinins. 6-(3-Methyl-2-butenylamino)purine, 6-(4-hydroxy-3-methyl-2-czs-butenyl-... 

The findings that inosinate was the end product of the pathway of de novo synthesis and that adenylate and guanylate were the first metabolites formed in the course of the incorporation into nucleic acids of adenine and guanine, were most consistent with interconversion at the ribonucleo-... 

There is little evidence regarding the cyclic operation of these reactions. One cycle may function in muscle where the extensive deamination of adenylate accompanying muscle function must be followed by its rapid resynthesis. McFall and Magasanik S3) have suggested that in cultured L cells, guanine is converted to adenine nucleotides for storage and then converted back to guanine nucleotides for incorporation into nucleic acids. 

In the above-mentioned experiment, labeled inosine, adenine, adenosine, and adenylic acid were incorporated into nucleic acid adenine to a greater extent than into nucleic acid guanine. This suggests that all the compounds, including inosine, may be related in their utilization by conversion to a common metabolite. 

Adenine is inert in the nucleoside phosphorylase systems of both mammalian tissues and microorganisms, but isotopically labeled adenine is effectively incorporated into nucleic acid purines, both in rats " and in yeast.This poses a question as to the possible role of nucleoside phosphorylase in polynucleotide synthesis. It has been suggested that hypoxanthine or guanine nucleosides (or nucleotides) are synthesized first. Then an exchange reaction with free adenine (or a derivative) might occur, For example, adenine might react with inosine to form adenosine and hypoxanthine. Some known exchange reactions are discussed below. 

The fate of other purine-ribose compounds was studied in the rat and it was found that C Mabeled adenosine (211) and adenylic acid (212) were utilized for the s3Tithesis of RNA adenine and guanine, but to a much smaller extent than adenine (191). Similarly, growing yeast utilized the purine base, adenine, far more readily than the corresponding nucleoside or nucleotide (195). It was believed that the ribose derivatives were poorly utilized because they were first cleaved to free adenine, which was incorporated subsequently into polynucleotides. It is curious that the attachment of ribose or a ribose pho hate moiety to adenine or guanine did not facilitate their incorporation into nucleic acids. In contrast, inosine, the ribonucleoside of hypoxantbine, was utilized considerably by the rat as a nucleic acid precursor (211) the corresponding deoxyriboside, deoxyinosine, was not (213). 

Seasonal variations in the metabolic fate of adenine nucleotides prelabelled with [8—1-4C] adenine were examined in leaf disks prepared at 1-month intervals, over the course of 1 year, from the shoots of tea plants (Camellia sinensis L. cv. Yabukita) which were growing under natural field conditions by Fujimori et al.33 Incorporation of radioactivity into nucleic acids and catabolites of purine nucleotides was found throughout the experimental period, but incorporation into theobromine and caffeine was found only in the young leaves harvested from April to June. Methy-lation of xanthosine, 7-methylxanthine, and theobromine was catalyzed by gel-filtered leaf extracts from young shoots (April to June), but the reactions could not be detected in extracts from leaves in which no synthesis of caffeine was observed in vivo. By contrast, the activity of 5-phosphoribosyl-1-pyrophosphate synthetase was still found in leaves harvested in July and August. 

The fact that purine bases could be used directly for nucleotide and nucleic acid i thesis was first established by the use of labeled compounds. Plentl and Schoenheimer (5) were not able to demonstrate incorporation of N- anine into nucleic acids of rat viscera in 1944, but Brown later was able to show that it was utilized by mice. (It is now known that guanine is degraded more rapidly in rats than in mice.) Brown and his colleagues (4, 5) also demonstrated the incorporation of N-adenine into nucleic acids, and between 1948 and 1954 numerous studies were made of the incorporation of N- or >KJ-labeled purines into nucleotides and nucleic acids (see reference g). 

Fortunately, my graduate research in biochemistry at Baylor College of Medicine, in Houston, involved studies on the mechanism of formic acid oxidation in animal tissues and on the incorporation of this one-carbon compound into nucleic acid components and their precursors. This provided me with experience in the use of isotopic tracers and the background in biochemical research which proved crucial, in later years, for unraveUng the intermediates and mechanisms of synthesis of purines and other compounds, when I discovered the prebiotic synthesis of adenine and other building blocks of nucleic acids, and a general pathway or method for the prebiotic formation of oligodeoxynucleotides and peptides. 

Amethopterin treatment not only increased formate incorporation into liver acid-soluble adenine compounds but also increased the incorporation of formate into nucleic acid and protein 202). It has been foimd that incorporation of formate-C into liver proteins and nucleic acids is also stimu-... 

The effect of 6-mercaptopurine on the incorporation of a number of C-labelled compounds into soluble purine nucleotides and into RNA and DNA has been studied in leukemia L1210, Ehrlich ascites carcinoma, and solid sarcoma 180. At a level of 6-mercaptopurine that markedly inhibited the incorporation of formate and glycine, the utilization of adenine or 2-aminoadenine was not affected. There was no inhibition of the incorporation of 5(or 4)-aminoimidazole-4(5)-carboxamide (AIC) into adenine derivatives and no marked or consistent inhibition of its incorporation into guanine derivatives. The conversion of AIC to purines in ascites cells was not inhibited at levels of 6-mercaptopurine 8-20 times those that produced 50 per cent or greater inhibition of de novo synthesis [292]. Furthermore, AIC reverses the inhibition of growth of S180 cells (AH/5) in culture by 6-mercaptopurine [293]. These results suggest that in all these systems, in vitro and in vivo, the principal site at which 6-mercaptopurine inhibits nucleic acid biosynthesis is prior to the formation of AIC, and that the interconversion of purine ribonucleotides (see below) is not the primary site of action [292]. Presumably, this early step is the conversion of PRPP to 5-phosphoribosylamine inhibited allosterically by 6-mercaptopurine ribonucleotide (feedback inhibition is not observed in cells that cannot convert 6-mercaptopurine to its ribonucleotide [244]. 

Base analogs such as 5-bromouracil and 2-aminopurine can be incorporated into DNA and are even more likely than normal nucleic acid bases to form transient tautomers that lead to transition mutations. 5-Bromouracil, an analog of thymine, normally pairs with adenine. However, the proportion of 5-bromouracil in the enol tautomer is higher than that of thymine because the bromine atom is more electronegative than is a methyl group on the C-5 atom. Thus, the incorporation of 5-bromouracil is especially likely to cause altered base-pairing in a subsequent round of DNA replication (Figure 27.42). 

Scott, J. L., Human leukocyte metabolism in vitro. I. Incorporation of adenine-8-C and formate-C into the nucleic acids of leukemic leukocytes. J. Clin. Invest. 41, 67-79 (1962). 

---