Deoxyribonucleoside triphosphate nucleotides

Deoxyribonucleoside triphosphate dependence All four required All four work, but single nucleotide will incorporate All four... 

DNA chain elongation is catalyzed by DNA polymerase III using 5 -deoxyribonucleoside triphosphates as substrates. The enzyme "proofreads" the newly synthesized DNA, removing terminal mismatched nucleotides with its 3 —>5 exonuclease activity. 

Gemcitabine is phosphorylated initially by the enzyme deoxycytidine kinase and then by other nucleoside kinases to the di- and triphosphate nucleotide forms, which then inhibit DNA synthesis. Inhibition is considered to result from two actions inhibition of ribonucleotide reductase by gemcitabine diphosphate, which reduces the level of deoxyribonucleoside triphosphates required for the synthesis of DNA and incorporation of gemcitabine triphosphate into DNA. Following incorporation of gemcitabine nucleotide, only one additional nucleotide can be added to the growing DNA strand, resulting in chain termination. 

The chemistry of the elongation reaction catalyzed by DNA polymerase I is shown in Figure 13. The enzyme catalyzes the nucleophilic attack of the 3 -0H terminus of the primer molecule on the a-phosphorus of the deoxyribonucleoside triphosphate to form a new phosphodiester bond with release of pyrophosphate. Elongation of the DNA chain proceeds in the 5 +3 direction at a rate of approximately ten nucleotides per second per molecule of DNA polymerase I. It is thought that the reaction is processive, in that many nucleotide units are added without release of the enzyme from the template. 

In the absence of deoxyribonucleoside triphosphates, DNA polymerases with associated 3 - 5 exonucleases would eventually degrade duplex DNA to mononucleotides. The replacement DNA synthesis method thus requires suf-ficiendy high levels of deoxyribonucleotides to maximize nucleotide poly-... 

Biotinylated dUTP can also be used to label DNA probes by a different method, namely random-primed labeling (4). The principle of this method is based on the reannealing of hexadeoxyribonucleotide primers, which have random specificity, to the denatured DNA strands. The DNA to be labeled has to be linearized and denatured before the strands are used as templates in the labeling reaction. The complementary strands are synthesized from the 3 OH termini of the reannealed hexanucleotides by the Klenow fragment of E. coli DNA polymerase I. The primers reanneal at random sites of the template strands, so that the synthesis of the complementary strands is primed at random sites. If one of the deoxyribonucleoside triphosphates present in the reaction mixture is labeled, the newly synthesized strands will become labeled by the incorporation of the labeled nucleotides. The end product of this reaction is a mixture of unlabeled (template) and labeled... 

Most of the carbon that flows through nucleotide synthetic pathways goes into ribonucleotide triphosphates (rNTPs - ATP, CTP, GTP, and UTP). A relatively small fraction is diverted to the synthesis of deoxyribonucleoside triphosphates (dNTPs). rNTPs are synthesized in excess of dNTPs because most cells contain 5-10 times as much RNA as DNA and because rNTPs have multiple metabolic roles, whereas dNTPs are used only to make DNA. 

In the dideoxy method a small piece of DNA called a primer, labeled at the 5 -end with is added to the restriction fragment whose sequence is to be determined. Next, the four 2 -deoxyribonucleoside triphosphates are added as well as DNA polymerase, the enzyme that adds nucleotides to a strand of DNA. In addition, a small amount of the 2, 3 -dideoxynucleoside triphosphate of one of the bases is added to the reaction mixture. A 2, 3 -dideoxynucleoside triphosphate has no OH groups at the 2 -and 3 -positions. 

E. coli has three DNA polymerases, Pol I, Pol II, and Pol III. Pol III is the major replicative enzyme (Table 13.1). All DNA polymerases that have been studied copy a DNA template strand in its 3 to 5 direction, producing a new strand in the 5 to 3 direction (Fig. 13.4). Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) serve as substrates for the addition of nucleotides to the growing chain. 

The DNA to be sequenced is mixed with a short oligonucleotide that serves as a primer for synthesis of the complementary strand. The primer is hydrogen-bonded toward the 3 end of the DNA to be sequenced. The DNA with primer is divided into four separate reaction mixtures. Each reaction mixture contains all four deoxyribonucleoside triphosphates (dNTPs), one of which is labeled to allow the newly synthesized fragments to be visualized by autoradiography or by fluorescence, as described in Section 13.1. In addition, each of the reaction mixtures contains one of the four ddNTPs. Synthesis of the chain is allowed to proceed in each of the four reaction mixtures. In each mixture, chain termination occurs at all possible sites for that nucleotide. 

The PCR process was first published by MuUis in 1985. It uses a mixture of the DNA to be duplicated, heat-stable polymerase, required by the polymerase, the four 2 -deoxyribonucleoside triphosphate building blocks (A,T,C,G), and the oligonucleotide primers (about 20 nucleotides long). There are three main steps in the process ... 

The basics of chemistry for the synthesis of DNA by the polymerization of deoxyribonucleoside triphosphates (dNTP) involve condensation of dNTPs or oligo-dNTP and dNTP monomer by phosphodiester linkages and maintenance of the chain elongation process to obtain the final DNA with specific numbers of nucleotides in specific sequences and appropriate base pairing through hydrogen bonds. 

The known in-vitro properties of DNA polymerizing enzymes do not provide an explanation of the in-vivo initiation of DNA synthesis (Kornberg, 1969 DeLucia and Cairns, 1969). Consider in-vitro polymerization of deoxyribonucleoside triphosphates by E, coli DNA polymerase. This enzyme catalyzes the addition of deoxyribonucleoside triphosphates to the 3 -hydroxyl terminus of a primer DNA. Such synthesis occurs only in the direction of 5 to 3 , and in all cases studied there is an absolute requirement for DNA template (Kornberg, 1969). The action of E, coli polymerase is illustrated in Figure 4. A DNA template must have an available 3 -hydroxyl terminated strand which can serve as a primer for the initiation of synthesis, and it is assumed that polymerase is bound to an area of the template strand near the 3 -end of the primer. The deoxynucleoside triphosphate is bound adjacent to the 3 -hydroxyl group of the terminal nucleotide to form a base pair with the template. When the correct base pair is formed the polymerase catalyzes a nucleophilic attack by the 3 -hydroxyl group of the primer on the a-phosphorus of the triphosphate. A phosphodiester bond is formed with the subsequent release of pyrophosphate (Fig. 4). 

The nucleotide patterns of cultured animal cells and ascites tumor cells are generally less vulnerable than those of tissues however, if washing is required, a medium capable of supporting energy metabolism should be employed. If cells are to be packed by centrifugation prior to extraction, cultures or ascitic fluids may first have to be cooled. Cells are extracted in the cold some procedures employ alternate freezing and thawing in the presence of acidic extractants. Extraction of cultured mouse embryo cells with 60% methanol for 16 hours at — 20 C has been employed in the analysis of deoxyribonucleoside triphosphates 9). 

Acid-soluble extracts can be assayed for certain nucleotides by enzymatic methods. Various assay procedures for the adenosine phosphates and the pyridine nucleotide coenzymes are available 22,33). Specific methods for the determination of picomole amounts of the four deoxyribonucleoside triphosphates have been reported recently 15,16). 

The ribonucleoside triphosphate reductase of L. leichmannii is an allosteric enzyme, the activity of which is modified in a complex manner by deoxyribonucleoside triphosphates 28, 29). Reduction of each of the four substrates is maximally stimulated by a particular deoxyribonucleoside triphosphate, which Beck 29) terms a prime effector. The data of Table 16-III illustrate the specific nature of the effector stimulation prime effectors are indicated by the italicized data. The effector-induced stimulation of reductase activity appears to be countered in particular ways by deoxyribonucleoside triphosphates for example, dTTP inhibits the dATP-acti-vated reduction of CTP. It has been speculated that these complicated positive and negative allosteric effects produced by nucleotides may constitute a mechanism for ensuring that surpluses or shortages in the production of deoxyribonucleotides do not occur in the cell 29). 

TLC has proved suitable for analysis of the products of enzymatic degradation of nucleotide coenzymes [38, 72] and of soluble ribonucleic acids [3] it has been demonstrated that enzymatic reactions can be carried out on layers of PEI-cellulose [72]. The biosynthesis of nucleosides [110], ribonucleoside- and deoxyribonucleoside triphosphates [52], diribonucleoside di-, tri- and tetraphosphates [76, 108, 111] and polynucleotides [62] has been followed with the help of TLC. 

Only deoxyribonucleoside triphosphates can serve as substrates. If one of the four nucleotides is missing, the reaction rate approaches zero. 

---