5 - -2 -deoxyuridines -triphosphate

Fig. 7. Schematic representation of the principle of TUNEL assay. The enzyme TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3 -OH ends of double- and single-stranded DNA. After Br-dUTP incorporation, DNA break sites are identified by an FITC-labeled anti-BrdU monoclonal antibody.

Another route to dUMP is the reduction of UDP to dUDP, followed by phosphorylation of dUDP to dUTP (or direct reduction of UTP to dUTP in some microorganisms). The dUTP is then hydrolyzed to dUMP. This circuitous route to dUMP is dictated by two considerations. First, the ribonucleotide reductase in most cells acts only on ribonu-cleoside diphosphates, probably because this permits better regulation of its activity. Second, cells contain a highly active deoxyuridine triphosphate diphosphohydrolase (dUT-Pase). It prevents the incorporation of dUTP into DNA by keeping intracellular levels of dUTP low by means of the reaction... 

Deoxyribose Deoxyuridine Deoxyuridylic acid deoxyuridine monophosphate (dUMP) Deoxyuridine diphosphate (dUDP) Deoxyuridine triphosphate (dUTP)... 

Williams MV, Winters T, Waddel KS. 1987. In vivo effects of mercury (II) on deoxyuridine triphosphate nucleotidohydrolase, DNA polymerase (alpha, beta), and uracil-DNA glycosylase activities in cultured human cells Relationship to DNA damage, DNA repair, and cytotoxicity. Mol Pharmacol 31 200-207. 

Hokaii S, Hasegawa M, Sakagishi Y, Kikuchi G (1987) Deoxyuridine triphosphate nucleotidohy-drolase activity and its correlation with multiplication of erythroid cells in rat spleen. Biochem Int 14 851-857... 

FU-treated cells, both fluorodeoxyuridine triphosphate (FdUTP) and deoxyuridine triphosphate (dUTP) (the substrate that accumulates behind the blocked TS reaction) incorporate into DNA in place of the depleted physiological nucleotide, TTP. Presumably, the incorporation ofdUTP and/or FdUTP into DNA activates the excision-repair process. This can lead to DNA strand breakage because DNA repair requires TTP, which is lacking because of TS inhibition. 5-FU incorporation into RNA also causes toxicity because of effects on both the processing and functions ofRNA. 

Bibenzocyclooctyne and bicyclo [6.1.0] non-4-yne-modified 2 -deoxyuridine triphosphates can be further employed for PCR reactions, with standard DNA polymerases allowing internal labeling of DNA with multiple strained alkyne residues [10]. Fluorescent labeling was demonstrated with azide-modified fluo-rophores [10]. 

Analogs of the natural deoxynucleoside triphosphates can be used for DNA synthesis, both in vivo and in vitro, but substitution of unnatural for natural deoxynucleotide must conform with the requirements for base-pairing in the Watson-Crick model of DNA. Thus with the purified enzyme system, thymine could be replaced by uracil or 5-bromouracil, 5-methyl- and 5-bromocytosine for cytosine, and hypoxanthine for guanine ( 6). Although chemically synthesized deoxyuridine triphosphate can be incorporated into DNA, there is apparently no kinase in nature which phosphorylates deoxyuridylate to the triphosphate stage. This may account for the absence of uracil nucleotides in DNA ( 6). 

Fluoro-2 -deoxyuridine has been extensively used in studies of the mechanism of action of thymidylate synthase, and 5-fluorouracil is an anticancer drug that has provided a lead to the development of others. The metabolism of 5-fluorouracil by the ascomycete fungus Nectria haematococca has been studied using F NMR (Parisot et al. 1991). a-Fluoro-P-alanine (2-fluoro-3-aminopropionate) was produced (Figure 10.27), while 5-fluorouridine-5 -mono-, di-, and triphosphate were found in acid extracts of the mycelia, and the 2 - and 3 -monophosphates were recovered from RNA. 

Fig. 24.4 Thymidylate synthase (TS) biochemical pathway. dUMP-deoxyuridine monophosphate, dTMP-deoxythymine monophosphate, dTTP-deoxythymine triphosphate.

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

Dideoxyuridine (ddU) is an antiviral agent that proved ineffective at controlling human immunodeficiency virus type 1 (HIV-1) infection in human T-cells. This ineffectiveness was ascribed to a lack of substrate affinity of ddU for cellular nucleoside kinases, which prevent it from being metabolized to the active 5 -triphosphate. To overcome this problem, bis[(pivaloyloxy)methyl] 2, 3 -dideoxyuridine 5 -monophosphate (9.41) was prepared and shown to be a membrane-permeable prodrug of 2, 3 -di-deoxyuridine 5 -monophosphate (ddUMP, 9.42) [93]. Indeed, human T-cell lines exposed to 9.41 rapidly formed the mono-, di-, and triphosphate of ddU, and antiviral activity was observed. This example again documents... 

Fig. 3. Metabolism of the fluoropyrimidines dTMP = deoxythymidine monophosphate, dUMP = deoxyuridine monophosphate, FdUDP = fluorodeoxyuridine diphosphate, FdUMP - fluoro-deoxyuridine monophosphate, FdUTP = fluorodeoxyuridine triphosphate, FU-DNA= fluorouracil-deoxyribonucleic acid, FUDP = fluorouracil diphosphate, FUMP = fluorouracil monophosphate, FU-RNA = fluorouracil-ribonucleic acid, FUTP = fluorouracil triphosphate.

Yoshioka A. Tanaka S, Hiraoka O, et al. Deoxyribonucleoside triphosphate imbalance. 5-fluoro-deoxyuridine-induced DNA double strand breaks in mouse FM3 A cells and the mechanism of cell death. J Biol Chem 1987 262 8235-8241. 

Trifluridine (Viroptic) is a fluorinated pyrimidine nucleoside that has in vitro activity against HSV-1 and HSV-2, vaccinia, and to a lesser extent, some adenoviruses. Activation of trifluridine requires its conversion to the 5 monophosphate form by cellular enzymes. Trifluridine monophosphate inhibits the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) by thymidylate synthetase. In addition, it competes with deoxythymidine triphosphate (dTTP) for incorporation by both viral and cellular DNA polymerases. Trifluridine-resistant mutants have been found to have alterations in thymidylate synthetase specificity. 

Fig. lA. Anabolic and catabolic pathways of 5-FU. DPD dihydropyrimidine dehydrogenase, DP di-hydropyrimidinase, pUP beta-ureidopropionase, UP uridine phosphorylase, OPRT orotate phospho-ribosyl transferase, UK uridine kinase, TP thymidine phosphorylase, TK thymidine kinase, RNR ribonucleotide reductase. The three active metabolites (shown in rectangles) are FdUMP (5-fluoro-2 -deoxyuridine 5 -monophosphate) inhibiting TS (thymidylate synthase), and FUTP (5-fluorouridine 5 -triphosphate) and FdUTP (5-fluoro 2 -deoxyuridine 5 -triphosphate) interfering with RNA and DNA, respectively. 

Fluorouracil (5-FU) is inactive in its parent form and requires activation via a complex series of enzymatic reactions to ribosyl and deoxyribosyl nucleotide metabolites. One of these metabolites, 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), forms a covalently ternary complex with the enzyme thymidylate synthase and the reduced folate 5,10-methylenetetrahydrofolate, a reaction critical for the de novo synthesis of thymidylate. This results in inhibition of DNA synthesis through "thymineless death." 5-FU is converted to 5-fluorouridine-5 -triphosphate (FUTP), which is then incorporated into RNA, where it interferes with RNA processing and mRNA translation. 5-FU is also converted to 5-fluorodeoxyuridine-5 -triphosphate (FdUTP), which can be incorporated into cellular DNA, resulting in inhibition of DNA synthesis and function. Thus, the cytotoxicity of 5-FU is thought to be the result of combined effects on both DNA- and RNA-mediated events. 

Fig. 31. Connection of 5-(3"-aminopropynyl)-2 -deoxyuridine-5 -triphosphate to the succinimide active ester arm of a ruthenium polypyridyl complex.

The full synthesis of two Fc-labeled bases 5-(3-ferrocenecarboxamidopropenyl-l) 2 -deoxyuridine 5 -triphosphate (Fcl-dUTP) and 5-(3-ferroceneacetamidopropenyl-... 

Sources of deoxyuridine in DNA include the presence of dUTP because dUMP (the substrate for thymidylate synthase) or dUDP (from ribonucleotide reductase action on UDP) are phosphorylated to the triphosphate. DNA polymerase recognizes these compounds as substrates. Another source is the deamination of deoxycytidine in DNA, promoted by a variety of compounds. If deoxyuridine is on a template strand of the DNA, it will direct the incorporation of an A in the newly made strand of DNA. This will convert a G-C pair to an A-T pair. 

Kornberg s work on the biosynthesis of deoxyribonucleic acid has shown that enzymes in Escherichia coli extracts catalyze the formation of the 5-triphosphates of 2-deoxyadenosine, 2-deoxyguanosine, 2-deoxycyti-dine, and thymidine from the corresponding monophosphates in the presence of adenosine 5-triphosphate, but fail to catalyze phosphorylation of deoxyuridine 5-phosphate this finding could explain why uracil is not a constituent of deoxyribonucleic acid. 

Cells that have been preincubated with deoxyuridine, then exposed to [ H] thymidine, incorporate little or none of the labeled material into DNA. This is because of both dilution of the labeled material in the larger intracellular pool of newly synthesized TMP and also inhibition of thymidylate kinase by thymidine triphosphate. 

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