DTTP synthesis

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

These enzymes use DNA as a template and the ribonucleotide substrates must be present in the nucleus, i.e. ATP, GTP, CTP and UTP. Similarly, for the synthesis of DNA, the deoxyribonucleotides dATP, dGTP, dCTP and dTTP must be present in the nucleus. In addition, since the ribonucleoside diphosphates are required for synthesis of deoxyribonucleotides, these diphosphates must also be present. The concentrations of these various nucleotides have not been measured in the nucleus but it may be assumed that the concentrations of the ribonucleotides will be similar in the nucleus to those in the cytosol. 

Figure 20.12 (a) Details of reaction catalysed by thymidylate synthase. Methylene FH4 represents N N methylene tetrahydro-folate (see Figure 15.2). (b) Reactions in the pathways in which either CDP or UDP gives rise to deoxythymidine monophosphate. Note that two processes can be involved in synthesis of deoxyuridine monophosphate. It is not known if one process dominates, but in (c) it is assumed that the pathway from CDP dominates formation of dTTP. (c) k summary of the reactions required for synthesis of deoxyribonucleotides required for DNA replication. 

Each new double helix is comprised of one strand that was part of the original molecule and one strand that is newly synthesized. Not surprisingly, this is a very simplistic description of a quite complex process, catalysed by enzymes known as DNA polymerases. The precursors for synthesis of the new chain are the nucleoside triphosphates, dATP, dGTP, dTTP, and dCTP. We have already met ATP when we considered anhydrides of phosphoric acid (see Box 7.25) these compounds are analogues of ATP, though the sugar is deoxyribose rather than ribose. 

Polymerase An enzyme which catalyses the synthesis of DNA using a single DNA strand as a template. The polymerase copies the template in the 5 -3 direction provided that sufficient quantities of free nucleotides, dATP and dTTP are present, [nih]... 

Removal of abnormal bases Abnormal bases, such as uracil, which can occur in DNA either by deamination of cytosine or improper incorporation of dUTP instead of dTTP during DNA synthesis, are recognized by specific glycosylases that hydrolytically cleave them from the deoxyribose-phosphate backbone of the strand. This leaves an apyrimidinic site (or apurinic, if a purine was removed), referred to as an AP-site. 

UTP, and CTP are needed for RNA synthesis and the 2 -deoxyribonucleotide triphosphates, dATP, dTTP, dGTP, and dCTP for DNA synthesis. In every case, the addition of activated monomer units to a growing polynucleotide chain is catalyzed by an enzyme that... 

Since DNA contains thymine (5-methyluracil) as a major base instead of uracil, the synthesis of thymidine monophosphate (dTMP, or thymidylate) is essential to provide dTTP (thymidine triphosphate), which is needed for DNA replication together with dATP, dGTP, and dCTP. 

The manner in which the reduction of ribonucleotides to deoxyribonucleotides is regulated has been studied with reductases from relatively few species. The enzymes from E. coli and from Novikoff s rat liver tumor have a complex pattern of inhibition and activation (fig. 23.25). ATP activates the reduction of both CDP and UDP. As dTTP is formed by metabolism of both dCDP and dUDP, it activates GDP reduction, and as dGTP accumulates, it activates ADP reduction. Finally, accumulation of dATP causes inhibition of the reduction of all substrates. This regulation is reinforced by dGTP inhibition of the reduction of GDP, UDP, and CDP and by dTTP inhibition of the reduction of the pyrimidine substrates. Because evidence suggests that ribonucleotide reductase may be the rate-limiting step in deoxyribonucleotide synthesis in at least some animal cells, these allosteric effects may be important in controlling deoxyribonucleotide synthesis. 

A second enzyme on the pathway to dTTP that is subject to allosteric control is deoxycytidylate deaminase, which supplies dUMP for thymidylate synthesis. The enzyme in mammalian cells, yeast, and bacteriophage T2-infected E. coli. is allosterically activated by dCTP (hydroxymethyl dCTP for the phage enzyme) and inhibited by dTTP. 

Fig. 1. DNA synthesis. In this schematic diagram, the incoming dTTP hydrogen bonds with the adenine on the template DNA strand and a 3 5 phosphodiester bond is formed, releasing pyrophosphate.

The regulation of ribonucleotide reductase is complex, with many feedback reactions used to keep the supplies of deoxynu-cleotides in balance. For example, dGTP and dTTP are feedback inhibitors of their own formation. Each is also an activator of the synthesis of the complementary nucleotide (dCDP or dADP), while dATP is an inhibitor of the reductions to make dADP, dCDP, dGDP, and dUDP. These control functions keep the supply of deoxynu-cleotides in balance, so that a roughly equivalent amount of each remains available for DNA synthesis. 

Thymidine is taken up by cells and rapidly converted to dTTP, the pool size of which is related to the extracellular thymidine concentration (see 12.1). At thymidine concentrations as low as 3 x 10-7M this leads to a measurable effect on the rate of DNA synthesis (Cooper et al., 1966). At concentrations above 1 mM inhibition of DNA synthesis is almost complete for some cell lines (Morris and Fischer, 1960 Xeros, 1962 Bootsma et al., 1964 ... 

Inhibition of DNA synthesis is brought about by the action of dTTP as an allosteric inhibitor of ribonucleotide reductase (Reichard et al., 1961 Moore and Hurlbert, 1966 Brown and Reichard, 1969 Rummer et al., 1978). This enzyme is responsible for reducing all four ribonucleoside diphosphates (NDP) to the corresponding de-oxyribonucleoside diphosphates (dNDP). It is subject to a complex allosteric control which has been most studied with the bacterial enzyme. Most studies with the mammalian enzyme show it to be similar to the bacterial enzyme (Fig.11.7). 

Hydroxyurea also inhibits DNA synthesis by its action on the M2 subunit of ribonucleotide reductase, but in this case it is the reduction of the purine nucleoside diphosphates which is inhibited and the pool of dTTP rises slightly (Turner et al., 1966 Adams and Lindsay, 1967 Krakoff et al., 1968 Adams et al., 1971 Skoog and Bjursell, 1974 Thelander et al., 1984). What prevents the pool rising dramatically is not clear, but some mechanism comes into play to reduce turnover of the dTTP pool (Nicander and Reichard, 1985). Its action is most satisfactorily reversed by changing the medium for drug free medium. 

Although there is obviously a through-put such that [3H]dTTP is being formed and removed by DNA synthesis it appears that the size of the radioactive deoxythymidylate pool is determined by the concentration of extracellular thymidine (Fig. 12.2). This may come about by a combination of forward promotion of thymidine kinase by its substrate and feedback inhibition by dTTP (Ives et al., 1963). The size of the pool remains constant once equilibrium has been reached until extracellular thymidine levels begin to fall. This will happen within a few hours when the concentration of extracellular thymidine is 10-6M and even at 10-5 M 10% may be utilised within... 

The endogenously synthesised dTTP dilutes the specific activity of the [3H]dTTP formed from the added [3H]thymidine. Thus on adding tritiated thymidine at 3 X 10 8M most of the DNA thymine is synthesised by the endogenous or de novo pathway, but when the [3H]thymidine concentration in the medium is raised to 0.3 mM it contributes 90% or more of the DNA thymine (Cleaver and Holford, 1965 Cooper et al., 1966 Cleaver, 1967). As the specific activity of [3H]dTTP is one of the factors which determine the amount of radioactivity incorporated into DNA (either total counts/min or grain counts) and as this varies (a) with external thymidine concentration and (b) with the state of the cells, the quantitative estimation of rates of DNA synthesis is full of pitfalls. 

The rate of incorporation of radioactivity is dependent on the specific activity of the [3H]dTTP which in the two previous methods has been assumed to be the same as that of the supplied [3H]thymi-dine. As the concentration of [3H] thymidine is increased from low values (i.e. less than 10-6M) the specific activity of the [3H]dTPP pool rises and so does the incorporation of radioactivity into DNA. This is at a time when the true rate of DNA synthesis remains unchanged. Thus the proporation of the dTTP pool which is radioactive, ([3H]dTTP(dTTP + [3H]dTTP)), is equal to the proportion of DNA thymine which is radioactive ([3H]thymine/total thymine). This equation may be rearranged to give ... 

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