ObjectivesIntegrity of both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) is affected by nutritional and environmental factors. Folate and B12 are water-soluble B-vitamins that act as cofactors in folate-mediated one-carbon metabolism (FOCM), a series of one-carbon transfer reactions that support several essential cell processes including nucleotide biosynthesis. Impairments in FOCM, such as folate or vitamin B12 deficiency, can disturb thymidylate (dTMP, the “T” base in DNA) synthesis and lead to uracil misincorporation to DNA. Both folate and B12 deficiency as well as genetic polymorphisms affecting FOCM function are associated with increased uracil accumulation and DNA damage in nDNA. However, dTMP synthesis occurs in multiple cellular compartments (cytosol, nucleus, and mitochondria), and it is not well defined how FOCM impairments affects mtDNA integrity and nucleotide pool balance between compartments. MethodsCurrently, we are using in vivo and in vitro experiments to assess nucleotide synthesis and uracil accumulation in a functional model of vitamin B-12 deficiency with decreased expression of methionine synthase (Mtr), one of two B12-requiring enzymes. Tissues from Mtr+/+,+/– mice and Mtr+/+,+/- mouse-derived cells were exposed to either folate-replete or folate-deplete conditions to explore the combined effects of folate and B12 deficiency on nucleotide synthesis capacity and integrity of both nDNA and mtDNA. ResultsWe have developed and validated a novel real-time PCR-based assay to quantify uracil misincorporation into mtDNA. In mouse liver, we observed a significant increase of uracil in mtDNA with decreased Mtr expression as well as an interaction between Mtr genotype and folate status. Mouse embryonic fibroblasts (MEFs) demonstrated perturbed dTMP synthesis and increased uracil accumulation in nDNA with decreased Mtr expression. ConclusionsThese data suggest that functional B12 deficiency, as modeled by decreased Mtr expression, disrupts nucleotide synthesis in more than one cellular compartment and increases uracil accumulation not only in nDNA, but also in mtDNA. Reduced Mtr expression may cause a redistribution of folates in the cytoplasm which impacts mitochondrial folate levels, and subsequently mitochondrial dTMP synthesis. Funding SourcesN/A.