Nucleotide incorporation pathway

Correct and incorrect nucleotide incorporation pathways in DNA polymerase b. Biochem. Biophys. Res. Commun., 350, 521-529. 

Thioguanine (6-TG) also inhibits several enzymes in the de novo purine nucleotide biosynthetic pathway. Various metabolic lesions result, including inhibition of purine nucleotide interconversion decrease in intracellular levels of guanine nucleotides, which leads to inhibition of glycoprotein synthesis interference with the formation of DNA and RNA and incorporation of thiopurine nucleotides into both DNA and RNA. 6-TG has a synergistic action when used together with cytarabine in the treatment of adult acute leukemia. 

The amino acid and nucleotide biosynthetic pathways make repeated use of the biological cofactors pyridoxal phosphate, tetrahydrofolate, and A-adenosylmethionine. Pyridoxal phosphate is required for transamination reactions involving glutamate and for other amino acid transformations. One-carbon transfers require S-adenosyhnethionine and tetrahydrofolate. Glutamine amidotransferases catalyze reactions that incorporate nitrogen derived from glutamine. 

Fig. 1. Kinetic pathway of nucleotide incorporation. The various complexes are indicated as mentioned in the text. kpol, the rate constant of the rate-limiting step, is indicated.

The rate-limiting step in the kinetic pathway of nucleotide incorporation is the conversion of the E p/t dNTP complex to the activated complex, E p/t dNTP (Step 3 in Fig. 1). This step is crucial in many respects. First, it is essential for the phosphoryl transfer reaction to occur. During the E p/t dNTP to E p/t dNTP transition, all the components of the active site are assembled and organized in a topological and geometrical arrangement that allows the enzyme to proceed with the chemical step (Step 4). Second, Step 3 plays a major role in the mechanism of discrimination between correct versus incorrect nucleotides. Interpretation of the kinetic measurements has led to the hypothesis that the E p/t dNTP... 

Translesion synthesis with DNA Pol of the A-acetyl-2-aminofluorene adduct of guanosine (88) is inefficient with templates containing (88). In the presence of the Revl protein, translesion synthesis occurs and dCTP is the major nucleotide incorporated opposite it, and studies with a mutant DNA Pol I gave similar results. Benzo[a]pyrene is a potent environmental carcinogen, which when metabolised leads to u t -benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide anti-BPDE). With dG, the major lesion is (+)-tra w-a h-B[a]P-A -dG, (89), and is usually repaired by the nucleotide excision repair (NER) pathway. The translesion synthesis past (89) has been examined with a number of polymerases. With human RNA Pol II, (89) is a block to synthesis, whilst DNA Pol k preferentially incorporated the correct nucleotide. In yeast cells, Pol induced a large number of mutations involving Pol p, whilst Pol p alone contributed to 1-3 deletions or insertions. The NER of (89) with UvrB proteins was also studied. ... 

Preliminary biosynthetic studies have shown that both cysteine and methionine are precursors of PSC, while cysteine but not methionine is also a precursor of DCS [6]. Methionine was also shown to supply both the sulfur and the S-methyl groups of PSC, suggesting that methionine is converted to sulfocholine which is incorporated into PSC by a pathway [6] analogous to the nucleotide (Kennedy) pathway for PC biosynthesis [7]. 

Antimetabolites are enzyme inhibitors (see p. 96) that selectively block metabolic pathways. The majority of clinically important cytostatic drugs act on nucleotide biosynthesis. Many of these are modified nucleobases or nucleotides that competitively inhibit their target enzymes (see p. 96). Many are also incorporated into the DNA, thereby preventing replication. 

When nucleoside analogs, such as cytosine arabinoside (AraC), azidothymidine (zidovudine orAZT), and dideoxyinosine (ddi), are converted into the corresponding nucleotides by salvage pathways, they can be Incorporated into nascent DNA strands by DNA polymerases. 

Discussing the biosynthetic pathways for amino acids and nucleotides together is a sound approach, not only because both classes of molecules contain nitrogen (which arises from common biological sources) but because the two sets of pathways are extensively intertwined, with several key intermediates in common. Certain amino acids or parts of amino acids are incorporated into the structure of purines and pyrimidines, and in one case part of a purine ring is incorporated into an amino acid (histidine). The two sets of pathways also share... 

The biosynthetic pathway to pyrimidine nucleotides is simpler than that for purine nucleotides, reflecting the simpler structure of the base. In contrast to the biosynthetic pathway for purine nucleotides, in the pyrimidine pathway the pyrimidine ring is constructed before ribose-5-phosphate is incorporated into the nucleotide. The first pyrimidine mononucleotide to be synthesized is orotidine-5 -monophosphate (OMP), and from this compound, pathways lead to nucleotides of uracil, cytosine, and thymine. OMP thus occupies a central role in pyrimidine nucleotide biosynthesis, somewhat analogous to the position of IMP in purine nucleotide biosynthesis. Like IMP, OMP is found only in low concentrations in cells and is not a constituent of RNA. 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

Phosphorus is abundant on Earth, both as an element (the llth-most abundant atom in Earth s crust) and as phosphate. Meteorites hold a variety of phosphate-containing minerals and some phosphide minerals.10 Scientists at the University of Arizona have recently suggested that Fe3P, the mineral schreibersite, leads to the formation of phosphate and phosphite when corroded in water. Although phosphorylation of alcohols was not demonstrated, mechanistic considerations suggest that it should be possible. It is noteworthy that a clear prebiotic pathway for the chemical incorporation of phosphate into RNA or DNA has not been found. No nucleosides (nucleobases joined to sugars) have been reported from meteorites. Nor has evidence been found in any meteorite of the presence of nucleosides or nucleotides (nucleosides attached to phosphates). That suggests that nucleic acids were first formed as products of metabolism. 

Mode of action Like acyclovir, ganciclovir is activated through conversion to the nucleoside triphosphate by viral and cellular enzymes, the actual pathway depending on the virus. Cytomegalovirus is deficient in thymidine kinase, and therefore forms the triphosphate by another route. The nucleotide competitively inhibits viral DNA polymerase and can be incorporated into the DNA to decrease the rate of chain elongation. 

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