Incorporation of Uracil into DNA

A required step for the in vivo incorporation of uracil into DNA is the... 

Reduced synthesis of thymidine (Section 10.3.3) may lead to incorporation of uracil into DNA, thus leading to genomic instability. 

Decreased methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), a reaction that requires N, N °-methyl-ene tetrahydrofolate as a coenzyme (see Fig. 40.5), leads to an increase in the intracellular dUTP/dTTP ratio. This change causes a significant increase in the incorporation of uracil into DNA. Although much of this uracil can be removed by DNA repair enzymes, the lack of available dTTP blocks the step of DNA repair catalyzed by DNA polymerase. The result is fragmentation of DNA as well as blockade of normal DNA rephcation. 

Mutation in the regulatory gene for deo (deoxyribose) operon. It allows more efficient uptake of large DNA fragments. It also allows growth with inosine as the sole carbon source dUTPase activity abolished. In combination with ung, it allows incorporation of uracil into DNA... 

DNA contains thymine rather than uracil, and the de novo pathway to thymine involves only deoxyribonu-cleotides. The immediate precursor of thymidylate (dTMP) is dUMP. In bacteria, the pathway to dUMP begins with formation of dUTP, either by deamination of dCTP or by phosphorylation of dUDP (Fig. 22-43). The dUTP is converted to dUMP by a dUTPase. The latter reaction must be efficient to keep dUTP pools low and prevent incorporation of uridylate into DNA. 

Acrylic acid causes toxicity by rapid polymerization in the presence of light, heat, and oxygen and thereby interfering with the incorporation of thymidine into DNA and uracil into RNA and inhibits protein synthesis. 

The dUTPase reaction is very important because the DNA polymerases cannot distinguish dUTP from TTP and catalyze significant incorporation of dUMP into DNA when dUTP is present. Incorporation of the base U into DNA is not deleterious because all cells contain a uracil... 

Experiments with animal cells have shown that the pyrimidine bases are much less effective DNA precursors than the corresponding deoxy-ribonucleosides, although the interpretation of such experiments is complicated by the rapid catabolism of uracil and thymine which takes place in liver. The incorporation of thymine into DNA in animals (14), or in in vitro systems (15) is slow and contrasts with the much more rapid incorporation of thymidine. 

Flucytosine is converted into the anti metabolite 5-fluorouracil that inhibits thymidilate synthetase, thereby disrupting DNA synthesis. It also interferes with protein synthesis by incorporation of fluorouracil into RNA in place of uracil. Although active against most Candida species, its spectrum of antifungal activity, overall, is narrow. Since resistance can develop rapidly it is usually coadministered with another agent and its main value is that it facilitates a reduction in the dose (and, presumably, the toxic effect) of amphotericin when co-prescribed in this way. The main adverse effects are marrow aplasia and hepatotoxicity. 

In addition to the methods described here, measurement of urinary ac-etamido p-aminohenzoyl glutamate will reflectfolate turnover (Section 10.2.3) and incorporation of uracil, instead of thymidine into the DNA in leukocytes or lymphocytes may provide a sensitive index of folate status. 

The nick-translation method was originally used to incorporate radiolabeled nucleotides into DNA probes. More recent work has shown that nucleotides labeled with biotin can also be incorporated using this method, to yield probes of equivalent sensitivity. Uracil and adenine nucleotides that have been labeled with biotin are shown in Figure 9.18. 

Consistent with the metabolic data, there is no dihydrofolate reductase/thymidylate synthase activity (75). Thymidine is salvaged by a phosphotransferase. Uracil PRTase, uridine phosphorylase, cytidine deaminase and uridine and cytidine phosphotransferases were found. The major pyrimidine salvaged is uracil via its PRTase. The lack of incorporation of salvaged uracil into DNA and the lack of thymidylate synthase indicates that this parasite cannot synthesize TMP from UMP. It is dependent on salvage for its thymidine requirements. This parasite possesses a hydroxyurea-resistant ribonucleotide reductase and can synthesize deoxycytidine nucleotides from cytidine nucleotides. 

Uracil mustard is not a uracil antagonist for E. coli Bu, which requires this nutrilite [479]. However, in rats with Walker 256 carcinosarcoma, incorporation of uracil-2- C into the RNA of intracellular tumour and spleen fractions is markedly inhibited [488]. UracU mustard also inhibits the incorporation of nucleic acid precursors into RNA and markedly inhibits DNA synthesis in tissues of A/J mice [489]. Inhibition of arginine incorporation into nuclear proteins was likewise noted [490]. 

In animal cells engaged in DNA synthesis, deoxyuridine can be incorporated without cleavage into DNA thymidylate, but phosphorolysis also occurs and the uracil so released will not have incorporation into DNA as a specific metabolic fate. Because of deamination, deoxycytidine may be converted to either of the pyrimidine nucleotides of DNA cleavage of the deamination product, deoxyuridine, liberates uracil which may enter the pathways of pyrimidine ribonucleotide metabolism. 

Uracil N-glycosylase activity abolished. Mutant permits incorporation of uracil residues into DNA. In mg host, the U residues in U-DNA are removed, leaving abasic sites Mutation in D-xylose isomerase, rendering the strain unable to utilize xylose... 

Fluorouracil (5-fluorouracil, 5-FU, Fig. 5) represents an early example of rational drag design in that it originated from the observation that tumor cells, especially from gut, incorporate radiolabeled uracil more efficiently into DNA than normal cells. 5-FU is a fluorinated pyrimidine analog that must be activated metabolically. In the cells 5-FU is converted to 5-fluoro-2>deoxyuridine-monophosphate (FdUMP). This metabolite inhibits thymidilate synthase which catalyses the conversion of uridylate (dUMP) to thymidilate (dTMP) whereby methylenetetrahydrofo-late plays the role of the carbon-donating cofactor. The reduced folate cofactor occupies an allosteric site of... 

Although FdUrd produces only DNA-medicated cyotoxicity, 5-FU can also be metabolized to fluorouracil monophosphate (FUMP) and ultimately to fluorouracil triphosphate (FUTP), which can be incorporated into RNA in place of uridine triphosphate (UTP). In other words, incorporation of 5-FU into RNA mimics uracil de novo synthesis and affects the production of ribosomal RNAs (rRNAs) (16,17). 5 -FU also affects several aspects of messenger RNA (mRNA) function, including transcription (18), translation (19), and slicing (20). 

Formation of dUMP in eukaryotes may occur by hydrolytic removal of phosphate from dUDP or from the conversions dCDP —> dCMP —> dUMP (steps k and V, Fig. 25-14). A more roundabout pathway is employed by E. coli dCDP —> dCTP —> dUTP —> dUMP (steps /c, /, and m, Fig. 25-14). One of the intermediates is dUTP. DNA polymerases are able to incorporate dUMP from this compound into polynucleotides to form uracil-containing DNA. Tire only reason that this does not happen extensively within cells is that dUTP is rapidly converted to dUMP by a pyrophosphatase (step m, Fig. 25-14). Tire uracil that is incorporated into DNA is later removed by a repair enzyme (Chapter 27). The presence of dUTP in DNA provides the basis for one of the most widely used methods of directed mutation of DNA (Chapter 26). 

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