Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. Marc Tini

Abstract

Thymine DNA glycosylase (TDG) is an essential DNA repair enzyme mediating excision of uracil and thymine mispaired with guanine within CpG contexts. Unrepaired, these lesions result in G:C to A:T transitions which are major contributors to genome instability. Interestingly, TDG interacts functionally with transcriptional regulators and participates in directed cytosine demethylation at promoters. TDG is subject to multiple post-translational modifications (PTM) and we undertook an analysis of how these regulate TDG function. Initially, we examined TDG regulation by small ubiquitin-like modifier (SUMO) and identified a novel SUMO binding motif (SBM1, residues 144-148). We hypothesized that SBM1, along with SBM2 (319-322), would facilitate non-covalent SUMO interactions upon conjugation of SUMO (sumoylation) to lysine 341, altering TDG conformation and function. Biochemical and cell based analyses supported our hypothesis, showing SUMO interactions allosterically regulate TDG protein-protein and substrate interactions, altering TDG subnuclear localization and enzymatic function. Furthermore, sumoylation drastically reduced acetylation of TDG occurring at lysines 70, 94, 95, and 98. Secondly, we examined TDG regulation by phosphorylation and demonstrated that serines 96 and 99 are phosphorylated by protein kinase C α in vivo. Biochemical analysis of covalently modified recombinant TDG showed that acetylation and phosphorylation of TDG are mutually exclusive and both are suppressed by TDG-DNA interactions. Furthermore, acetylated TDG did not interact stably with DNA or efficiently excise thymine from G:T mispairs, while phosphorylated TDG was indistinguishable from unmodified protein. Lastly, we examined TDG regulation in aging cells. Immunostaining showed TDG redistributed from nucleus to cytoplasm in aged cells. Interestingly, treatment with histone deacetylase inhibitors resulted in similar redistribution and immunoblotting indicated that an increase in TDG modification consistent with sumoylation or monoubiquitination had occurred. Similar results were obtained by exposing cells to oxidative stress. Analysis of a sumoylation-minus mutant of TDG identified sumoylation as an important regulator of TDG localization. Interestingly, we found extensive colocalization of TDG with sites of active transcription which was reduced by phorbol ester treatments which surprisingly promoted entry into heterochromatic regions from which TDG is generally excluded. Together, these findings suggest that TDG function may be regulated by PTM, consequently affecting genome stability and expression.

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