DNA contains thymine rather than uracil, and the de novo pathway to thymine involves only deoxyribonucleotides. The immediate precursor of thymidylate (dTMP) is dUMP. In bacteria, the pathway to dUMP begins with formation of dUTP, either by deamination of dCTP or by phosphorylation of dUDP. The dUTP is converted to dUMP by a dUTPase. The latter reaction must be efficient to keep dUTP pools low and prevent incorporation of uridylate into DN

Conversion of dUMP to dTMP is catalyzed by thymidylate synthase. A one-carbon unit at the hydroxymethyl (–CH 2 OH) oxidation level is transferred from N 5 , N 10 -methylenetetrahydrofolate to dUMP, then reduced to a methyl group.

The reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate-requiring reactions.

The dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase—a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihydrofolate reductase reside on a single, bifunctional protein.