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Start studying Chapter 12 DNA Replication

For three of the four nucleotides—ADP, CDP, and GDP—the conversion to the dNTP involves simply a phosphorylation of the rNDP reductase product, catalyzed by nucleoside diphosphate kinase. In most cells, this enzyme is very active, and all known forms of the enzyme have very low specificity. Thus, the enzyme catalyzes the reversible transfer of the g-phosphate of any common rNTP or dNTP to the phosphate at the b-position of any common rNDP or dNDP. The equilibrium constant for each reaction catalyzed is close to unity. Thus, the direction in which a nucleoside diphosphate kinase-catalyzed reaction occurs in vivo depends on the concentrations of substrates and products. Because ATP is almost always the most abundant intracellular nucleoside triphosphate, most such reactions involve the ATP-dependent conversion of a ribo- or deoxyribonucleoside diphosphate to the corresponding triphosphate.

kinds of deoxyribonucleoside triphosphate ..

Much of what is known about pathways of dNTP synthesis in bacterial and animal cells came from investigations of the biosynthesis of unusual modified nucleotides in infection by certain bacteriophage (11). T-even bacteriophages of E. coli contain 5-hydroxymethylcytosine completely substituted for cytosine; many hydroxymethyldeoxycytidylate (HM-dCMP) residues in phage DNA are further modified by glycosidic links through the hydroxymethyl group to one or two glucose residues. These modifications occur through the action of phage-encoded enzymes that catalyze reactions comparable to those in cellular metabolism (Fig. 4). For example, the hydroxymethylation reaction is carried out by an enzyme, dCMP hydroxymethylase, that transfers a single-carbon group from methylenetetrahydrofolate to C-5 of dCMP, much as thymidylate synthase modifies C-5 of the pyrimidine dUMP (12); in fact, T-even phages encode a thymidylate synthase that displays significant amino acid sequence homology with dCMP hydroxymethylase. Not shown in Figure 4 is the involvement of glucosylation in the transfer of glucose to hydroxymethyl groups of HM-dCMP residues after their incorporation into DNA.

Deoxynucleoside [1-thio]triphosphates prevent …

. Now the deoxyribose-1-phosphate can react with another base in anucleoside phosphorylase-catalyzed reaction proceeding in the reverse direction, eg,Thymidine can then become a nucleotide via the thymidine kinase reaction leading to dTMP. Although this process can significantly change relative dNTP pool sizes, it does not involve net deoxyribonucleotide synthesis; rather, it involves redistribution of the deoxyribosyl units linked to purine and pyrimidine bases.

By contrast, dAMP and dGMP kinases play roles only in nucleotide salvage reactions because the de novo pathways lead directly from ribonucleoside diphosphate to deoxyribonucleoside diphosphate to deoxyribonucleoside triphosphate.

of intracellular deoxyribonucleoside triphosphate ..

Nucleotide kinase-catalyzed reactions are readily reversible. Whereas these enzymes have long been considered to participate primarily in dNTP synthesis, there is now good evidence that nucleotide kinases function in both directions in vivo and that they can participate in dNTP degradation (13). For example, when DNA replication was blocked in mammalian cells with aphidicolin, intracellular dNTPs were degraded, and deoxyribonucleosides were excreted into the medium in significant amounts (13, 14), with little change in the steady-state pool size of the four dNTPs. The implication of these findings is that dNTP pool sizes are controlled not only at the level of synthesis but are also regulated by catabolism.

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:

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DNA with modified deoxyribonucleoside triphosphate using ..

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).

pyrimidine 2'-deoxyribonucleoside 5'-triphosphate, ..

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.

SparkNotes: DNA Replication and Repair: Terms

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.

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