Folate mediated one-carbon (1C) metabolism is required for the de novo synthesis of purines, thymidylate, and the remethylation of homocysteine to methionine. Perturbations in folate mediated 1C metabolism are associated with pathologies and developmental defects including cardiovascular disease, cancers, and neural tube defects. Disruption of thymidylate biosynthesis is associated with genome instability resulting from the misincorporation of uracil into DNA. The de novo dTMP pathway consists of serine hydroxymethyltransferase, (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase (DHFR). Serine hydroxymethyltransferase 1 (SHMT1) expression is limiting for de novo thymidylate synthesis. Whereas the current literature describes nucleotide biosynthesis occurring in the cytoplasm, the results of these studies demonstrate that de novo dTMP biosynthesis is unique in that it occurs in the nucleus and mitochondria. The entire de novo thymidylate biosynthesis pathway is shown to undergo SUMO dependent nuclear translocation during S-phase. A novel isoform of SHMT, termed SHMT2[alpha], was identified which provides functional redundancy with SHMT1 for nuclear de novo thymidylate biosynthesis and accounts for about 25% of nuclear SHMT activity. SHMT1 protein levels are shown to be regulated by the ubiquitin system where an interplay between SUMOylation and ubiquitination on SHMT1 signals proteasomal degradation, nuclear export, and mediates stability within the nucleus and cytosol. Nuclear thymidylate biosynthesis is coupled with the formation of multi-enzyme complexes associated with the nuclear lamina. SHMT1 serves as scaffold protein and is essential for metabolic complex formation. The enzymes for de novo dTMP synthesis were found to be associated with PCNA and enriched at origins of replication. The nuclear compartmentation of de novo dTMP synthesis led to the hypothesis that there is a requirement for dTMP synthesis within the mitochondria. Purified mitochondria were capable of de novo thymidylate biosynthesis. A novel isoform of DHFR previously thought to be a pseudogene called DHFR-like protein 1 (DHFRL1) was identified that is essential for mitochondrial thymidylate biosynthesis. In cells lacking mitochondrial SHMT activity (SHMT2) uracil levels in mtDNA were increased by 40%. These data support a role for de novo dTMP synthesis within the nucleus and mitochondria that supports DNA replication and repair and limits uracil misincorporation.