Deoxynucleotide synthesis

Both dihydrofolate reductase and thymidylate synthase reactions are targets for anticancer chemotherapy. Cancer is basically a disease of uncontrolled cell replication, and an essential part of cell replication is DNA synthesis. This means that a requirement exists for deoxynu-cleotide synthesis for growth. Inhibition of deoxynucleotide synthesis should inhibit the growth of cancer cells. 

The best-studied enzymes to date that contain glycyl radicals are pyruvate formate-lyase (PFL) and a ribonucleoside triphosphate reductase (ARR), both isolated from anaerobically growing E. coli. These enzymes play central roles in the anaerobic metabolism of the bacterium. The first catalyzes the reversible formation of acetyl-CoA and formate from pyruvate and coenzyme A, while the second is responsible for synthesizing the deoxyribonucleotide monomers of the polymer DNA. It is intriguing to note that formate, a product of the PFL reaction, is a substrate for the ARR. It supplies the reducing equivalents needed for each round of deoxynucleotide synthesis. ... 

FIG. 6.1 Purine deoxynucleotide synthesis pathways. For identity of enzymes see legend to Fig. 6.1. 

Anabaena T-1 and spinach chloroplast Tm are the most abundant thioredoxins in vivo. They exhibit approximately 50% sequence homology to thioredoxins from other nonphotosynthetic organisms (9). They can be reduced by NADPH and the flavoprotein-type reductase (10). Presumably they have intracellular functions which are common to thioredoxins in all living organisms, such as deoxynucleotide synthesis and sulfate metabolism. 

In the diester method a deoxynucleoside-5 -monophosphate is condensed with the 3 -OH group of a deoxynucleotide to produce a 3, 5 -phosphodiester. This is illustrated by a general method for dinucleotide synthesis developed by H.G. Khorana (K.L. Agarwal, 1976). One N-... 

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

Purine Synthesis Purine Salvage Deoxynucleotides Purine Degradation... 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to be phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduction of the ribose to the 2 -deoxyri-bose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled by reduction with NADPH by the enzyme thioredoxin reductase. 

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. 

Regulation of the synthesis of the deoxynucleotides to ensure equal concentrations of all four nucleotides... 

An extract from a paper by P. Reichard (1988) illustrates the importance of ensuring the normal balance of all the deoxynucleotides prior to DNA synthesis. 

This system ensures that the cdck is either completely active, to facilitate sufficient enzyme synthesis that the deoxynucleotides... 

De novo synthesis of purines and pyrimidines yields the monophosphates IMP and UMP, respectively (see p. 188). All other nucleotides and deoxynucleotides are synthesized from these two precursors. An overview of the pathways involved is presented here further details are given on p. 417. Nucleotide synthesis by recycling of bases (the salvage pathway) is discussed on p. 186. 

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