Deoxy thymidine-5 -triphosphate

Cells that are synthesizing DNA mnst also be able to synthesize deoxy thymidine triphosphate (dTTP). The key step in the synthesis is the conversion of dUMP to dTMP via thymidylate synthetase. The reaction reqnires a sonrce of N, -methylene tetrahydrofolate to provide the methyl gronp. In this reaction the tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate is rednced to tetrahydrofolate via dihydrofolate reductase so more methylene If, A °-tetrahydrofolate is made from serine in a reaction that is catalyzed by serine hydroxymethyltransferase. These three reactions, which are essential for the formation of dTMP, are shown in Fig. 14-20. 

The de novo pathway to 2 -deoxythymidine monophosphate (dTMP) synthesis first requires the use of dUMP (2 -deoxyuridine-5 -monophosphate) from the metabolism of either UDP or CDP (cytidine diphosphate). The hydrolysis of dUTP (2 -deoxyuridine-5 -triphosphate) to dUMP and subsequent methylation at C-5 by the action of thymidylate synthase, using A, A i°-methylenetetrahydrofolate (THF) as the methyl donor, generate dTMP (Figure 6.54). The latter is subsequently phosphorylated to deoxy-thymidine triphosphate (dTTP) used in DNA synthesis and repair. 

Huang P, Farquhar D, Plunkett W. Selective action of 3 -azido-3 -deoxy-thymidine 5 -triphosphate on viral reverse transcriptases and human DNA polymerases. J Biol Chem 1990 265 11914-11918. 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Closely related to ATP, and present in biological systems, are a number of other nucleoside triphosphates and their corresponding diphosphates and monophosphates. These are uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and thymidine triphosphate (TTP), all of which have about the same free energy of hydrolysis as ATP (11.29). In addition there are the deoxy nucleoside triphosphates (Chapter 10.4). 

Fig. 14. Effect of 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FdUMP) on the synthesis of RNA and DNA. 5-FU is incorporated into RNA whereas 5-FdUMP inhibits thymidilate synthetase competitively and blocks DNA synthesis. The conversion of thymidine (dTr) into thymidilate (dTMP) by means of thymidine kinase is only a subsidary routine to thymidilate which can become important, however, under certain conditions such as in developing pollen (page 185). U = uridine triphosphate, Tr = thymidine, TMP = thymidine monophosphate = thymidilate, TDP = thymidine diphosphate, TTP = thymidine triphosphate, 5-F = 5-fluoro, d = deoxy, 1 = thymidine kinase, 2 = thymidilate synthetase.

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

Separation of dTTP, dTDP-D-glucose, dTDP, and dTMP (where dTMP, dTDP, and dTTP are the deoxy forms of thymidine mono-, di-, and triphosphate, respectively) occurred on a Supelcosil, LC-SAX column (4.6 mm x 250 mm). The chromatogram was developed at room temperature with a 20 mL linear gradient from 50 to 400 mM potassium phosphate buffer (pH... 

Acyclovir is an acyclic guanosine derivative that requires three phosphorylation steps for activation. Conversion to the monophosphate is carried out by the virus thymidine kinase, and conversion to the di- and triphosphate is carried out by host kineases. The active nucleotide triphosphate inhibits viral replication by competing with the viral deoxy-GTP for the viral DNA polymerase. Thus, acyclovir triphosphate is inserted into the growing viral DNA, leading to irreversible chain termination. Reduced susceptibility to acyclovir is due to altered viral thymidine kinase. Because the MOA of ganciclovir is similar to that of acyclovir, HSV that is resistant to acyclovir is also resistant to ganciclovir. 

Novel 2, 3 -deoxy fluorescent 3 -C-branched thymidine 5 -triphosphate analogues have been prepared and employed as potential terminators in DNA-sequencing reactions. The amino-nucleotide derivatives (105), (106) have been dye-labelled at the aliphatic amino-group with the oxazine dye JA242. ... 

As regards modified intemucleotidic links, bis(deoxynucleosidyl)-long-chain alkyl triphosphates have been reported, along with thymidine linked via either 0-3 or 0-5 to 0-6 of methyl a-D-glucopyranoside through a hydrophobic phosphotriester. A known derivative of 3 -deoxy-3 -C-formylthymidine (Vol. 28, p. 277-8) has been converted into a phosphoramidite which was used to make oligomers with a five-atom phosphate link, as in 178. ... 

Dicarboxylic acid 5 -monoesters of thymidine and of 5-(2-thienyl)-2 -deoxy-uridine have been prepared as triphosphate mimics. The ester of d4T with citric acid has also been made with the same objective, and more lipophilic bioisosteres 237 and the AZT equivalent have been reported, these being designed to mimic the conformation of the nucleoside triphosphate when complexed to a metal ion. Neither of these last two classes were effective, since all the activity against HIV could be shown to be due to hydrolysis to the parent nucleoside. 

Generally, UV absorption spectra can be utilized to determine the family to which an unknown base, nucleoside, or nucleotide belongs. Indeed, the chro-mophore of the nucleosides or nucleotides is typical of the purine or pyrimidine structvu-e, with different maximum absorption wavelengths in the adenosine, guanosine, cytosine, or thymidine series. For example, it is possible to differentiate readily the UV absorption spectra of guanosine (G), G-monophosphate (GMP), deoxy GMP (dGMP), G-diphosphate (GDP), and G-triphosphate (GTP), from those of adenosine (A), AMP, dAMP, ADR, and ATP. 

K. Ganeshaguru and A.V. Hoffbrand. The effect of deoxy-uridine, vitamin B12, folate and alcohol on the uptake of thymidine and on the deoxynucleoside triphosphate concentrations in normal and megaloblastic cells. Brit. J. Haematol. 40 29, 1978. 

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