Investigation into TDG Germline Variant-Mediated 5-Fluorouracil Cellular Sensitivity

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Thymine DNA Glycosylase (TDG) is a DNA glycosylase involved in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, but is also active on 5-flurouracil (5-FU) paired with adenine and guanine. There is a variant of TDG found in 10% of the global population where residue Gly199 is mutated to Ser. Gly199 is responsible for stabilizing the flipped abasic nucleotide in the active site pocket and mutation of this residue could affect the function of this protein. Interestingly, we have shown that cells expressing G199S have increased sensitivity to treatment with chemotherapeutic agent 5-FU compared to WT. Biochemical studies indicate G199S binds more tightly to an abasic product with a 4-fold lower Kd than WT. Additionally, expression of G199S in cells leads to an increase in 5-FU-induced single-strand DNA breaks compared to WT, as well as persistent double-strand breaks after treatment with 5-FU and recovery in a drug-free media for up to 48hrs. Together, these
results suggest G199S binds more tightly to its abasic product /in vivo/ preventing efficient downstream processing of the BER intermediate and
eventually leading to DNA break formation. I plan to further elucidate the cellular mechanism contributing to this increased sensitivity in hopes of understanding the role of G199S in 5-FU-directed toxicity.