DNA thymine

Nucleic acids are polymeric nucleotides in which phosphate esters link ribose or deoxyribose molecules through the C OH of one and the C OH of the other. In rna, the aglycone nitrogen bases are cytosine, adenine, guanine, and uracil. In dna, thymine replaces uracil. The rna polymer is like that of dna, except that a CH, replaces the OH group on C of the ribose ring. 

The endogenously synthesised dTTP dilutes the specific activity of the [3H]dTTP formed from the added [3H]thymidine. Thus on adding tritiated thymidine at 3 X 10 8M most of the DNA thymine is synthesised by the endogenous or de novo pathway, but when the [3H]thymidine concentration in the medium is raised to 0.3 mM it contributes 90% or more of the DNA thymine (Cleaver and Holford, 1965 Cooper et al., 1966 Cleaver, 1967). As the specific activity of [3H]dTTP is one of the factors which determine the amount of radioactivity incorporated into DNA (either total counts/min or grain counts) and as this varies (a) with external thymidine concentration and (b) with the state of the cells, the quantitative estimation of rates of DNA synthesis is full of pitfalls. 

Make allowance for the contribution of endogenous dTTP to DNA thymine. 

By measuring the incorporation of thymidine into DNA from [3H]thymidine supplied at different concentrations to mouse L cells, Cleaver (1967) was able to show that, at about 10-5M thymidine, incorporation reached a plateau, and a similar observation has been made for CHO cells (Fig. 12.3). This has been interpreted as showing that at this concentration the contribution of endogenous dTTP to DNA thymine is negligible. Care must be taken, however, that at... 

The rate of incorporation of radioactivity is dependent on the specific activity of the [3H]dTTP which in the two previous methods has been assumed to be the same as that of the supplied [3H]thymi-dine. As the concentration of [3H] thymidine is increased from low values (i.e. less than 10-6M) the specific activity of the [3H]dTPP pool rises and so does the incorporation of radioactivity into DNA. This is at a time when the true rate of DNA synthesis remains unchanged. Thus the proporation of the dTTP pool which is radioactive, ([3H]dTTP(dTTP + [3H]dTTP)), is equal to the proportion of DNA thymine which is radioactive ([3H]thymine/total thymine). This equation may be rearranged to give ... 

Wittes and Kidwell (1973) have described a kinetic approach to measuring the pool size of dTTP which involves growing cells continuously in 10/iM tritiated thymidine which they found to label about 20% of the DNA thymine residues. They calculated that at this concentration 106 L929 cells growing in suspension would convert 9.5 pmol extracellular thymidine into dTTP per min during S-phase. 

The key to DNA s functioning is its double-helical structure with complementary bases on the two strands. The bases form hydrogen bonds to each other, as shown in Fig. 22.38. Note that the structures of cytosine and guanine make them perfect partners for hydrogen bonding, and they are always found as pairs on the two strands of DNA. Thymine and adenine form similar hydrogen-bonding pairs. 

Heterocyclic compounds find prominent application in pharmaceutical and agricultural products, and they are also critical in biological processes. For example, the heterocyclic compound thymine is an important component of DNA. Thymine is also identified in lUPAC nomenclature as 5-methylpyrimidine-2,4(1 H,3H)-dione. 

A thymine dimer is a covalent linked between adjacent two thymine bases within a strand of DNA. Thymine dimers arise from ultraviolet-light induced formation of covalent bonds, as shown in Figure... 

Kanvah S, Schuster GB. One-electron oxidation of DNA thymine versus guanine reactivity. Org Biomol Chem. 2010 8 1340-1343. 

Mukherjee KL, Heidelberger C (1962) Studies of fluorinated pyrimidines. XV. Inhibition of the incorporation of formate-C14 into DNA thymine of ehrlich ascites carcinoma cells by 5-fluoro-2 -deoxyuridine-5 -monophosphate and related compounds. Cancer Res 22 815-822... 

However, for adenine, guanine, and uracil, the dominant route of anabolism is by way of their ribonucleotide derivatives and traffic along the deoxyribosidic route is not ordinarily significant. Because cytosine is not a substrate for nucleoside phosphorylases, incorporation by the phos-phorylase-kinase route is not possible for this base. The other pyrimidine base of DNA, thymine, is poorly anabolized by both animal and bacterial cells, in spite of the fact that most cells possess thymidine phosphorylase, the action of which is readily reversible. This suggests that ordinarily cellular supplies of deoxyribose 1-phosphate are not available for base anabolism. Experiments are cited below in which it was demonstrated that a significant contribution to the biogenesis of deoxyribose of DNA in E. colt cells did not occur by a route other than ribonucleotide reduction. 

Prior to the definitive experiments of Friedkin and Komberg (see below), early tracer studies with orotate and with nucleoside derivatives of uracil and cytosine had demonstrated that the pyrimidine ring of DNA thymine could be derived from these compounds. In addition, it had become apparent that the methyl group of thymine was derived from one-carbon units at the oxidation level of formaldehyde rather than at the oxidation level of formate formaldehyde and the hydroxymethyl group... 

It was found in early studies that the folic acid analogues, methotrexate (amethopterin) and aminopterin very effectively blocked the incorporation of labeled deoxyuridine and of labeled formate into DNA thymine however, the incorporation of thymidine was not blocked. It was apparent, therefore, that the analogues interfered with the introduction of the methyl group into thymine, a process known to involve H -folate. When it became established that the antifolic agents were exceedingly potent inhibitors of tetrahydrofolate dehydrogenase (see Chapter 5), the mechanism of their inhibition of DNA synthesis was apparent. 

N -Deoxyribosides of cytosine and thymine were prepared biosyntheti-cally from the DNA of E. colt which had been allowed to grow on an N -containing medium (319). When isotopic deoxycjrtidine was injected into rats, it was not incorporated into RNA pyrimidines, which indicated that the transformation of cytidine to deoxycytidine was not reversible (330). However, labeled deoxycytidine served as a precursor of both DNA thymine and cytosine, while labeled thymidine was found solely in thymine. Thus, the conversion of deoxycytidine to DNA thymine was another irreversible step. 

N DNA cytosine —deoxycytidine DNA thymine -thymidine Fig. 19. Utilization of pyrimidine nucleosides. 

The methylation of the pyrimidine nucleus by one-carbon compounds was in accord with the earlier suggestion that thymine formation in the rat proceeded by direct conversion of C3rtidine or a closely related structure to thymidine (317). Further agreement came from studies in rabbit bone marrow and in Ehrlich ascites tumor cells in vitro, where it was demonstrated that both c rtidine and deoxycytidine (and to a lesser extent uridine and deoxyuridine) stimulated the incorporation of formate-C into DNA thymine (338). 

Serrano-P rez, J., Gonzalez-Ramirez, 1., Goto, P., Serrano-Andrds, L., Merchan, M. (2008d). Theoretical insight into the intrinsic ultrafast formation of cyclobutane pyrimidine dimers in UV-irradiated DNA Thymine versus cytosine. The Journal of Physical Chemistry B, 112,14096-14098. 

Antony, J., Medvedev, D.M., and Stuchebrukhov, A.A., Theoretical study of electron transfer between the photolyase catalytic cofactor FADH and DNA thymine dimer, /. Am. Chem. Soc., 122, 1057, 2000. 

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