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The Synthesis and Degradation of Nucleotides

A little bit of searching on-line will show you that the metabolic pathways for the pyrimidines CTP, GTP and UTP are extensively covered! There is a thymidine triphosphate, but strictly speaking it is dTTP, deoxythymidine triphosphate. Thymidine triphosphate is required for the synthesis of DNA. dTTP is not made by the de novo pyrimidine synthesis pathway. e.g., in E.coli dTTP is made after UDP or CDP is converted to dUDP or dCDP (which can then be deaminated to dUTP) by action of the enzyme ribonucleoside diphosphate reductase (sometimes abbreviated as rNDPase). dUTP is converted to dUMP (by dUTPase), dUMP to dTMP (by thymidylate synthase), and dTMP is converted to TDP and dTTP by kinases for use in DNA synthesis. There are other ways (i.e., thymidine kinase salvage pathway for dTMP) for its recycling.

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).

10/12/2017 · Answer to The synthesis of dTTP appears to be energetically wasteful

Purine and Pyrimidine Metabolism - PURINES AND …

However, if the ratio of dUTP to dTTP is still elevated, this re-synthesis may again incorporate uracil instead of thymine.

In the thymidylate synthase reaction, the transferred methylene group must be reduced to the methyl level, and the electron pair that brings this reduction about comes from the reduced pteridine ring of 5,10-methylenetetrahydrofolate. The coenzyme, therefore, loses both its methylene group and an electron pair, leading to dihydrofolate. Transformation of the coenzyme for reuse involves, first, its reduction to tetrahydrofolate by dihydrofolate reductase and, next, transfer of a single-carbon group to the pteridine ring, usually catalyzed by serine transhydroxymethylase. The stoichiometric requirement for the folate cofactor in the thymidylate synthase reaction probably explains the selective toxicity of dihydrofolate reductase inhibitors toward proliferating cells (3). Such inhibitors include Methotrexate, widely used in cancer chemotherapy, and Trimethoprim, an antibacterial agent that specifically inhibits dihydrofolate reductases of prokaryotic origin(see Aminopterin). Proliferating cells have a continuous requirement for dTTP synthesis, to sustain DNA replication. The greater the flux rate through thymidylate synthase in vivo, the more rapidly tetrahydrofolate pools will be depleted after administration of a dihydrofolate reductase inhibitor and, hence, the greater will be the sensitivity of those cells toward the growth-inhibiting or lethal effects of blockage of dihydrofolate reductase.

Figure 3. dCMP deaminase as a branch point between dCTP and dTTP synthesis. Allosteric modifiers of dCMP deaminase are shown, as is the regulation of salvage synthetic pathways. Enzyme 1, ribonucleoside diphosphate reductase; 2, nucleoside diphosphate kinase; 3, dCMP kinase (possibly); 4, dUTPase; 5, dCMP deaminase; 6, dCMP kinase; 7, thymidylate synthase; 8, thymidylate kinase; 9, deoxycytidine kinase; 10, thymidine kinase.

Nucleotide Metabolism: Nucleic Acid Synthesis

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.

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Moreover, our own research has shown that, in larvae of the fruit fly Drosophila melanogaster, the ratio of dUTP to dTTP is regulated in an unusual manner: in all tissues that will not be needed in the adult insect, there are much lower levels of the enzyme that breaks down dUTP and generates a precursor for dTTP production. Consequently, significant amounts of uracil are incorporated into these tissues during DNA synthesis.

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Toyobo Life Science Department - Modifing Enzyme ReverTra Ace-


Antibiotic Resistance and Evolution - The Emperor Has …

If this strict regulation is perturbed and the ratio of dUTP to dTTP rises, the amount of uracil that is incorrectly incorporated into DNA also increases. The repair system – which, unlike DNA polymerases, can distinguish uracil from thymine – then attempts to cut out the uracil with the help of uracil-DNA glycosylase and to re-synthesise the DNA, which involves temporarily cleaving (cutting) the DNA backbone. However, if the ratio of dUTP to dTTP is still elevated, this re-synthesis may again incorporate uracil instead of thymine. This cycle eventually leads to DNA strand breaks and chromosome fragmentation, when these temporary cuts in the DNA happen one after the other and too close to each other (see ). This results in a specific type of programmed cell death, called thymine-less cell death.

Standard PCR protocol | Sigma-Aldrich

When DNA is synthesised, the DNA polymerase enzymes (which catalyse the synthesis) cannot discriminate between thymine and uracil. They only check whether the hydrogen bonds form correctly, i.e. whether the base pairs are matched properly. To these enzymes, it does not matter whether thymine or uracil binds to adenine. Normally, the amounts of deoxyuridine triphosphate (dUTP, a source of uracil) in the cell are kept very low compared to levels of deoxythymidine triphosphate (dTTP, a thymine source), preventing uracil incorporation during DNA synthesis.

DNA Structure - What is Life HOME

For salvage of deoxyribonucleoside monophosphates released by intracellular DNA degradation, the deoxyribonucleoside monophosphate kinases play the key roles. Animal cells contain four such enzymes, each specific for one deoxyribonucleotide, ie, dAMP kinase, dCMP kinase, dGMP kinase, and dTMP kinase . The enzyme phosphorylating dAMP acts also on AMP and is the well-known adenylate kinase, or myokinase. dCMP kinase acts also on UMP, and dTMP kinase acts also on dUMP. dTMP kinase is involved also in de novo dTTP synthesis, as shown in Figure 1. dCMP kinase may also play a role in de novo dNTP synthesis. The enzyme converting dCDP (produced by ribonucleotide reductase) to dCMP, en route to dUMP and dTMP, has still not been identified. Since nucleotide kinases all have equilibrium constants close to 1, it is quite possible that the role of dCMP kinase is to carry out the synthesis of dCMP:

Sanger sequencing - an overview | ScienceDirect Topics

The process of thymine-less cell death can be deliberately exploited in the treatment of cancer. Because cancer cells proliferate at such a high rate compared to normal cells, they synthesise a greater amount of DNA per given time period and therefore require large amounts of dUTP. By raising the ratio of dUTP to dTTP, these cancer cells can be selectively targeted and eliminated.

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