Folates are a family of enzyme cofactors that transfer one-carbon methyl groups in a network of connected metabolic pathways known as folate-mediated one-carbon metabolism (FOCM). FOCM generates purines, methionine, thymidylate (dTMP), and formate for cellular processes and is tightly regulated and compartmentalized in the cell. Impairments in FOCM have been associated with pathologies including neural tube birth defects (NTDs), cancers, neurodegeneration, and cognitive decline. The overall goal of this dissertation research was to identify mechanisms of impaired FOCM that contribute to disease, focusing on NTDs. NTDs are birth defects of the brain and spinal cord that occur due to incomplete neural tube closure. Approximately 70% of all human NTDs are folate-responsive, and show a polygenic and environmental component of risk, however the mechanism whereby folate prevents NTDs is not established. Serine hydroxymethyltransferase (SHMT1) disruption in mice leads to impaired de novo thymidylate synthesis, increased uracil in DNA, and folate-responsive NTDs. This implicates impaired de novo thymidylate synthesis in the pathogenesis of folate-responsive NTDs. However, it is not currently known how nuclear translocation of SHMT1 and its role in scaffolding the metabolic complex to the nuclear lamina and DNA replication machinery is required for folate-dependent NTD prevention or how dysregulated nucleotide pools, uracil misincorporation, and subsequent base excision repair (BER) processes contribute to this pathology. To investigate this, we used mouse and cell models with impairments in SHMT1, uracil DNA glycosylase (a BER enzyme), deoxythymidylate kinase (dTYMK), and lamin A. In the SHMT1 models, our results showed that the nuclear roles of SHMT1 in folate cofactor binding, metabolic complex scaffolding, and catalytic activity all contribute to the pathology of folate and SHMT1 -dependent NTDs in mouse models. Mouse models sensitized to NTDs by SHMT1 disruption combined with impaired BER showed a significantly lower penetrance of NTDs compared to SHMT1 models with no disruptions in BER. This implicates repair of uracil in DNA instead of its accumulation as the causal factor behind folate-responsive NTDs in mice. Together, these findings implicate DNA repair processes and dysregulated de novo dTMP biosynthesis in the polygenic and environmental pathology of NTDs.