Deoxyribonucleoside triphosphates

The reduction of ribonucleoside triphosphates by various dithiols which are capable of intramolecular cyclization on oxidation (dihydrolipoate, dithioerythritol, dithiothreitol) yields 2 -deoxyribonucleoside triphosphates. These reactions also require 5-deoxyadenosylcorrinoids. 

Figure 36-14. The initiation of DNA synthesis upon a primer of RNA and the subsequent attachment of the second deoxyribonucleoside triphosphate.

During the S phase, mammahan cells contain greater quantities of DNA polymerase than during the nonsynthetic phases of the cell cycle. Furthermore, those eiKymes responsible for formation of the substrates for DNA synthesis—ie, deoxyribonucleoside triphosphates—are also increased in activity, and their activity will diminish following the synthetic phase until the reappearance of the signal for renewed DNA... 

DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

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

Hirota Y, Yoshioka A, Tanaka S, et al. Imbalance of deoxyribonucleoside triphosphates, DNA doublestrand breaks and cell death caused by 2-chlorodeoxy adenosine in mouse FM3 A cells. Cancer Res 1989 49 915-919. 

Yoshioka A. Tanaka S, Hiraoka O, et al. Deoxyribonucleoside triphosphate imbalance. 5-fluoro-deoxyuridine-induced DNA double strand breaks in mouse FM3 A cells and the mechanism of cell death. J Biol Chem 1987 262 8235-8241. 

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. 

The Sanger dideoxynucleoside method of sequencing DNA. (a) A suitable template is chosen, and the primer is chosen so that DNA synthesis begins at the point of interest. The primer is radioactively labeled. In addition to the template-primer complex the reaction mixture contains all four radioactive deoxyribonucleoside triphosphates and small amounts of a single dideoxynucleoside triphosphate. The dideoxy compound serves as a chain terminator. 

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. 

Chain termination An incubation mixture is set up containing the single-stranded DNA template, method the primer, DNA polymerase I and all four deoxyribonucleoside triphosphates... 

DNA POLYMERASE Any of several enzymes that catalyze the formation of DNA from deoxyribonucleoside triphosphates, using one strand of DNA as a template. 

Synchrony at the Gl/S-interphase has been accomplished by interfering with the synthesis of one or more deoxyribonucleoside triphosphate, which are required for DNA synthesis while allowing other cellular procedures such as synthesis of RNA and protein to proceed. 

DNA repair is best studied in a system where the background levels of replication are low. Suitable systems are cultures which have come to rest at high density, or unstimulated lymphocyte preparations where less than 1% of the cells are in S-phase. The low levels of replicative incorporation can be further repressed by 1-2 mM hydroxyurea which selectively inhibits replication (Cleaver, 1969b). This selective effect may simply be a result of the very small pools of deoxyribonucleoside triphosphates required for repair. 

P]-Iabelled nucleoside triphosphates—Radiochemical Centre, Amersham, Bucks. U.K. or New England Nuclear, 2, New Road, Southampton, U.K. Deoxyribonucleoside triphosphates—Boehringer, P.-L. 

The gene encoding ppk2 (ppk2) was identified from the amino acid sequence of the purified protein. It encodes a protein of 357 amino acids with a molecular mass of 40.8 kDa. Both of the polyphosphate kinases ppkl and ppk2 may be involved in regulation of the level of ribonucleoside triphosphates and deoxyribonucleoside triphosphates that modulate cell division and survival in the stationary phase (Ishige et al., 2002). 

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-... 

Using double-stranded DNA with one or more single-strand discontinuities (nicks or breaks in the phosphodiester backbone), DNA polymerase 1 can extend the available 3 -OH termini in the presence of the four deoxyribonucleoside triphosphates. The reaction requires the concerted action of both the 5 3 ... 

The common denominators of RNRs are radical chemistry involving redox active cysteines (see sections 3.1 and 3.3) and with few exceptions an allosteric control mediated by deoxyribonucleoside triphosphates (dNTPs) and ATP (Reichard, 1997). Yet, several other characteristics distinguish the three RNR classes (Table 1) from each other ... 

A 2 deoxyribonucleoside triphosphate (dNTP) A 2, 3 "dideoxyribonucleoside triphosphate (ddNTP) ... 

The substrates for DNA synthesis are a 3 primed site and deoxyribonucleoside triphosphate (dNTP) (see Fig. 1). DNA polymerase catalyzes a phosphoryl transfer reaction that adds a dNMP moiety to a 3 terminus of an existing DNA strand, releasing pyrophosphate (Fig. la). The reaction is catalyzed exclusively by two metal ions (e.g., Mg++) (11, 12). Metal ion A extracts a proton from the DNA primer 3 terminal hydroxyl group to produce a oxyanion nucleophile, which attacks... 

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... 

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