Deoxyuridine phosphorylation

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

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... 

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. 

Takemura M, Yamamoto T, Kitagawa M, Taya Y, Akiyama T, Asahara H, Linn S, Suzuki S, Tamai K, Yoshida S (2001) Stimulation of DNA polymerase a activity by Cdk2-phosphorylated Rb protein. Biochem Biophys Res Commun 282 984-990 Tsukamoto I, Taniguchi Y, Miyoshi M, Kojo S (1991) Purification and characterization of thymidine kinase from regenerating rat liver. Biochim Biophys Acta 1079 348-352 VUpo JA (1983) Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes. Med Biol 61 54—58... 

Pyrazomycin. Pyrazomycin (11), 3-(l-p-D-ribofuranosyl)-4-hydroxypyrazole 5-carboxamide, is isolated from S. Candidas (1—4,9,10). The incorporation of [2-13C]acetate and [1- and U-14C]glutamate into the four contiguous carbons of pyrazomycin has been reported (11,12). Pyrazomycin 5 -phosphate inhibits orotidylic acid decarboxylase. Pyrazomycin inhibits adenosine phosphorylation and decreases the incorporation of deoxyuridine into DNA of Novikoff hepatoma cells in culture. It also inhibits the growth of tumor cells and the cytopathic effects of vaccinia, herpes simplex, vesicular stomatitis, Newcasde disease, measles, Sindbis, polio, hepatitis A, and coxsackie viruses (13,14). The inhibitory action of (11) on viral multiplication is reversed by uridine. 

Dihydro-5-aZathymidine. 5,6-Dihydro-5-azathymidine [57350-36-4] (177), C9H15N305, contains the x-triazine ring and is isolated from the culture filtrates of S. platensis var. clarensis (4). The inhibition of vital replication by (177) can be reversed by either thymidine, deoxyuridine, or deoxycytidine. Compound (177) protects mice following intracerebral inoculation with HSV-1 and appears to inhibit the phosphorylation of thymidine rather than its incorporation into DNA. Resistance to (177) by E. coli appears to result from a change in thymidine phosphorylase. 

FU, floxuridine (5-fluoro-2 -deoxyuridine, or 5-FUdR), aTK/idoxuridine (5-iodo-deoxyuridine) (see Chapter 49). lododeoxyuridine behaves as an analog of thymidine, and its primary biological action results from its phosphorylation and ultimate incorporation into DNA in place of thymidylate. In 5-FU, the smaller fluorine allows the molecule to mimic uracil biochemically. However, the fluorine-carbon bond is much tighter than that of C—H and prevents the methyla-tion of the 5 position of 5-FU by TS. Instead, in the presence of the physiological cofactor 5,... 

This deoxyribonucleoside prodrug (Fig. 42.26) is bioconverted via 2 -deoxyuridine kinase-mediated phosphorylation to the same active 5-fluoro-dUMP structure generated in the multistep biotransformation of... 

The synthetic pirimidine, 5-fluouracil (5-FU), is used in the treatment of a variety of epithelial tumors including, colorectal, breast cancer, ovarian, head, and neck cancers. 5-FU enters tumor cells by a facilitated nucleobase transporter and is converted into active metabolites (5 -fluoro-2 -deoxyuridine) by a complex metabolic pathway that interferes with both DNA and RNA syntheses. Of particular interest is the conversion of 5-FU to 5 -fluoro-2 -deoxyur-idine (5-FUdR) and subsequent phosphorylation by thymidine kinase to the active metabolite 5 -fluoro-2 -DUMP (5-FdUMP). Unfortunately, the response rate of patients receiving 5-FU-based chemotherapy is relatively poor, varying between 10 and 30%. To further evaluate, 5-FU labeled with F (5- FU) has been used in PET studies to understand the factors behind this strong interindividual variability. As 5- FU is biochemically identical to 5-FU, PET has been used for quantitative pharmacokinetic measurements of 5- FU in human and animal models. 

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. 

Metabolism of the pyrimidine deoxyribonucleotides is more complex because, in addition to transfer of phosphoryl groups, deamination and methylation reactions occur at this level. Specifically, the thymidine phosphates are derived by methylation of deoxyuridylate, and the latter may be derived from the deoxycytidine phosphates by way of deoxycytidylate deaminase. The deoxycytidine phosphates are not formed by amination of deoxyuridine phosphates, but are derived entirely from the cytidine phosphates by enzymatic reduction (Chapter 16). 

As seen in the diagram above, both deoxycytidine and deoxyuridine may be converted to deox3ruridylate. Deamination of the former by cyti-dine deaminase yields deoxyuridine, which is phosphorylated by thymidine kinase (see Chapter 14). Cytidine deaminase is responsible for the deamination of both cytidine and deoxycytidine (see Chapter 12) the enzyme may be under regulatory control in that it is inhibited by dTTP 18). 

These results are consistent with anabolism studies with cell cultures. A close analog of BrVdUrd, (E)-5-(2-iodovinyl)-2 -deoxyuridine, was efficiently converted to its monophosphate derivative in cells infected with both HSV-1 and HSV-2. However, a diphosphate (or triphosphate) could only be detected with extracts of cells infected with HSV-1 . The inefficient phosphorylation of BrVdUMP by the dThd-dTMP kinase from HSV-2 correlates well with the observation that replication of HSV-2 is at least 100-fold less sensitive to BrVdUrd than is that of HSV-1 and suggests a cause-and-effect relationship. The sensitivities of the two viruses to acyclovir are about equal. ... 

Because of the rapid synthesis of DNA during differentiation, lymphoid cells would be expected to be particularly sensitive to abnormalities in the regulation of the nucleoside pools that serve as precursors for DNA synthesis. The synthesis of thymidine triphosphate is of particular interest because of its specificity for DNA synthesis. The rate limiting step in the de novo pathway to dUMP involves conversion of dUPM to a dTMP by the folate requiring enzyme thymidylate synthetase, vitro incorporation of 3H-thymidine and 3n-deoxyuridine is reduced in bone marrow cultures from folic acid deficient rats (Sakamato and Takaku, 1973). Thymidylate synthetase activity was elevated when assayed in the presence of adequate cofactor (5,10-methylene THF). A salvage pathway also exists for the direct phosphorylation of thymidine by thymidine kinase. This enzyme can exist in two forms (Ellims et al., ... 

Deoxyuridine is also phosphorylated by extracts of E. coli, but at only one-third the rate of thymidine phosphorylation (83). Purification of E. coli extracts has revealed the presence of a kinase for each of the deoxyribo-nucleotides found in DNA (108). The conversion of uridine to uridine 5 -phosphate with ATP as phosphorylating agent has been demonstrated in yeast (83) and fiver (109). 

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). 

Azacvtidine - 5-Azacytidine inhibits the incorporation of tri-tiated thymidine or deoxyadenosine into DNA to a greater extent than it does tritiated uridine into RNA. The inhibition can be nullified by cyti-dine, or by uridine, but not by deoxycytidine or deoxyuridine, 5-Azacyti-dine inhibits the S phase (DNA synthesis) of the cell cycle. The activity of uridine kinase in mouse leukemia cells resistant to 5-azauridlne is reduced to ca. 507. of that in the sensitive cells.l-0 ll- This enzyme is responsible for the initial phosphorylation step of uridine, cytidlne, and... 

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