Open Access Thesis
Date of Award
Nutrition and Food Science
Background: The DNA base excision repair (BER) pathway is responsible for processing of genomic uracil lesions however, in some tissue types the excisional and gap-filling steps performed by UNG2 and POLβ, respectively, are impaired by folate deficiency in human and murine models in vitro. Genomic uracil damage can be acquired by inadequate conversion of uracil to thymine nucleotide precursors resulting from insufficient folate cofactors, or through activation induced cytosine deaminase (AID) activity during antibody diversification in B-cells in the context of adaptive immunity. The immunoglobulin (Ig) diversification methods in B-cells depend on the coordinated interaction between AID and UNG2, and as such, their analogous transcriptional regulation has been established. However, AID-induced uracil lesions at non-Ig loci have been found to be associated with lymphomagenesis, suggesting a functional BER deficit. Therefore, the objective of this research is to establish the tissue specific effect of folate deficiency on BER enzymes in B-lymphoblastoids, and to examine the functional coordination between AID expression and BER inducibility during moderate folate deficiency in B-cells in vitro. Methods: Human B-lymphoblastoid cells were cultured in 12nM and standard folate media concentration for 21 days. Intracellular folate concentration measured by microbiological assay, doubling time, and genetic expression of UNG, POLβ, and AID analyzed by RT-qPCR was completed with culture samples taken every 3-4 days. Results: Within 10 days in moderate folate deficient media, intracellular folate concentration was decreased by 93%, and cell proliferation was reduced by 80%. Expression of UNG was significantly downregulated by 16% at day 3 of folate deficiency, and by 44% at day 21. AID mRNA levels were significantly increased by 25% by day 10, demonstrating an inverse relationship between UNG2 and AID gene expression. Conclusion: Tissue specific downregulation of BER in folate deficient B-lymphoblastoids results in a functional imbalance between AID and uracil repair capacity, possibly increasing the risk of oncogenic transition mutations resulting from unrepaired AID-induced genomic uracil.
Zanley, Elizabeth, "Imbalance Of Uracil Dna Glycosylase And Activation-Induced Cytidine Deaminase Expression In Folate Depleted Human Lymphoblastoids" (2019). Wayne State University Theses. 729.