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There are two distinct pathways for dTTP synthesis

There are two major pathways controlling dTTP synthesis in cells, a de novo and a salvage pathway

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The Synthesis and Degradation of Nucleotides

AB - Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-xg)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-xg)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.

Purine and Pyrimidine Metabolism - EHSL

The most widespread pathway, shown in Figure 3, involves deamination at the monophosphate level. In many vertebrate cell lines, this is the predominant, often exclusive, pathway leading to dUMP (4). It is not clear why the in vivo flux rate from UDP to dUDP is so low, particularly when the activities of ribonucleotide reductase on CDP and UDP are regulated virtually identically as shown by assays of the purified enzyme in vitro (5). Whatever the reason, dCMP deaminase is an important metabolic branch point between routes to dTTP and dCTP. Allosteric regulation of this enzyme—activation by dCTP and inhibition by dTTP—ensures that these two dNTPs are produced at relative rates commensurate with their need for DNA synthesis. dCMP deaminase is not essential for cell viability, at least as determined in cell culture systems. However, mutant cells lacking dCMP deaminase have abnormally high dCTP pools (1, 6), and the resultant increase in the [dCTP]/[dTTP] pool ratio often brings about a mutator phenotype, in which the dCTP pool expansion stimulates its incorporation opposite template nucleotides other than dGMP (1).

Nucleotide Metabolism: Nucleic Acid Synthesis

20/10/2010 · Synthesis of dTTP by the de novo pathway takes a convoluted pathway from dUDP to dUTP to dUMP. The last reaction is catalyzed dUTPase. 5.

Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-xg)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-xg)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.

N2 - Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-xg)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-xg)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.

Cyclic adenosine monophosphate - Wikipedia

The de novo pathway to dTTP synthesis first requires the use of dUMP from the metabolism of either UDP or CDP.

Rhamnose, a naturally occurring deoxy-hexose, is found in the L- rather than the D-configuration assumed by most other sugars. In Bacteria, plants and fungi, rhamnose is a common component of the cell wall –, and was also recently found in viruses . At present, two pathways for synthesizing nucleotide-activated rhamnose are known. In Bacteria, RmlA, RmlB, RmlC and RmlD act sequentially to convert glucose-1-phosphate and deoxy-thymidine triphosphate (dTTP) into thymidine diphosphate (dTDP)-rhamnose , . Specifically, RmlA, the first enzyme of the pathway, is a glucose-1-phosphate thymidylyltransferase that combines thymidine monophosphate with glucose-1-phosphate to create dTDP-glucose. RmlB, a dTDP-glucose-4,6-dehydratase, then catalyzes the oxidation and dehydration of dTDP-glucose to form dTDP-4-keto 6-deoxy-glucose. RmlC, a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase, next performs a double epimerization at the C3 and C5 positions of the sugar. Finally, RmlD, a dTDP-4-dehydrorhamnose reductase, catalyzes the last step of the pathway, namely reduction of the C4 keto group of the sugar to yield dTDP-rhamnose. In plants, uridine diphosphate (UDP)-rhamnose rather than dTDP-rhamnose is generated by RHM (UDP-L-rhamnose synthase), a single polypeptide that contains all of the enzymatic activities required . Here, UDP-glucose is converted to UDP-4-keto-6-deoxy-glucose by an enzymatic activity similar to bacterial RmlB. Next, and in contrast to the bacterial process, whereby RmlC and RmlD operate sequentially to generate dTDP-rhamnose, plants instead rely on nucleotide-rhamnose synthase/epimerase-reductase, a bifunctional enzyme mediating both the epimerization and reduction reactions that lead to the biosynthesis of UDP-rhamnose –. More recently, the same pathway was shown to catalyze UDP-rhamnose biogenesis in large DNA viruses . The two pathways for nucleotide-activated rhamnose biosynthesis are depicted in .

The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.

Inosinic acid or inosine monophosphate (IMP) is a nucleoside monophosphate
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of dTTP pool size by the APC/C pathway during ..

As noted in Salvage pathways to nucleotide biosynthesis, deoxyribonucleotide salvage pathways involving uptake of extracellular precursors primarily use deoxyribonucleoside kinases. Human cells contain four such enzymes of varying specificities, two located in the cytosol and two in mitochondria, whereas other organisms, such as E. coli, contain thymidine kinase as the only deoxyribonucleoside kinase. Thymidine kinase has received particularly intensive study, partly because of the mechanism of its cell cycle regulation (9) but largely because the enzyme is so useful as a means for incorporating radiolabel into DNA. For reasons still not clear, thymidine competes extremely effectively with the de novo synthetic pathway to dTTP such that, in many animal cell systems, radiolabeled thymidine is incorporated into DNA at full specific activity, often bypassing substantial endogenous pools generated by de novo synthesis (10). One popular experimental organism for which this does not work is yeast; fungi lack thymidine kinase. Investigators have circumvented this difficulty, however, by designing yeast strains that are permeable to dTMP, strains for which exogenous dTMP can be used as a labeled DNA precursor.

SYNTHESIS OF PYRIMIDINE RIBONUCLEOTIDES

Regulation of cellular dTTP pools is essential for faithful replication of nuclear and mitochondrial DNA (mtDNA), with both depletion and expansion of the pool affecting DNA integrity and human health (). Depletion of de novo dTMP synthesis results in deoxyuridine misincorporation into nuclear DNA leading to genome instability (), whereas depletion of mitochondrial dTTP pools, due to mitochondrial deoxyribonucleoside kinases, deoxyguanosine kinase, or thymidine kinase 2 (TK2) mutations are associated with mtDNA-depletion syndromes and severe mitochondrial dysfunction in humans (). Elevations in dTTP pools, as observed in mitochondrial neurogastrointestinal encephalomyopathy (), an autosomal recessive disease resulting from decreased levels of cytoplasmic thymidine phosphorylase, results in mtDNA depletion (), deletions (), and site-specific point mutations ().

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