Synthesis of Thymidine Nucleotides

The dUTPase reaction is very important because the DNA polymerases cannot distinguish dUTP from TTP and catalyze significant incorporation of dUMP into DNA when dUTP is present. Incorporation of the base U into DNA is not deleterious because all cells contain a uracil 

Pathway for TTP synthesis. The key intermediate is dUTP, which is converted to dUMP by dUTPase. 

Because there is only one pathway for synthesis of TMP, it can be used to synthesize radioactively labeled DNA or to inhibit DNA synthesis selectively. In bacterial thymidylate synthase mutants (thy ), DNA synthesis is not possible without added thymine or thymidine, both of which can be utilized by salvage pathways. Neither thymine nor thymidine engages in synthetic reactions other than production of TMP, so radioactive thymine or thymidine can serve as unique precursors for synthesis of radioactive DNA. The radioactivity usually is in the methyl group since this group is not metabolized, the appearance of radioactivity in any other compound is avoided. 

Fluoropyrimidines are catabolized by enzymes that normally participate in the breakdown of endogeneous 

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Synthesis of Thymidine nucleotides first requires deoxyribonucleotide synthesis. The enzyme responsible for this step is Ribonucleotide Reductase. This enzyme acts on oxynucleotides in their diphosphate form. Thioredoxin, a small protein, is oxidized as the 2 hydroxyl group on the ribose ring is reduced. Oxidized Thioredoxin (S-S) is then reduced by FADH2 and NADPH. The products are the respective deoxynucleotide diphosphates which are further phosphorylated and then used for DNA synthesis. 

To avoid incorporating uridines into DNA, cells have developed a rather a simple mechanism. The enzyme, dUXPase, converts dUTP (a substrate for incorporation into DNA) to dUMP (not a substrate for incorporation into DNA), and also provides a route to synthesis of thymidine nucleotides because the dUMP, in turn, is converted first to dTMP then to dTTP (Figure 22.17). 

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