Date of Award
Doctor of Philosophy (PhD)
Genetics and Biochemistry
Dr. Weiguo Cao
Dr. F. Alex Feltus
Dr. Lukasz Kozubowski
Dr. Yanzhang Wei
Uracil-DNA glycosylase (UDG) superfamily, which consists of several groups of enzymes that recognize the damaged DNA bases and initiate the base excision repair (BER) pathway, is most important in dealing with DNA deamination and other base modifications. Thymine DNA glycosylase (TDG), which belongs to family 2 in the UDG superfamily, is able to specifically recognize and cleave the 5-methylcytosine (mC) oxidative derivatives including 5-formylcytosine (fC), 5-carboxylcytosine (caC), 5-hydromethyluracil (hmU) caused by active demethylation or DNA damage. My dissertation work is mainly focused on the fC and caC glycosylase activity within UDG superfamily. Chapter 1 is a general introduction to the UDG superfamily enzymes and DNA demethylation pathway. Chapter 2 presents a glycosylase activity screening within UDG superfamily. We extensively investigated an untraditional family 3 SMUG2 from Pedobacter heparinus to understand its specificity and catalytic mechanism in removing fC and caC. Phe SMUG2 has family 2 TDGs’ unique features, but still keeps most of structural features of family 3 SMUG1. In chapter 3, we conducted detailed biochemical and structural analyses of zebrafish TDG1 and TDG2 from family 2. A special mutant in motif 1 of zTDG2 has also been identified that can convert its glycosylase pattern to be more like zTDG1. Chapter 4 is an extended study of motif 1 of TDG from Scleropages formosus. Through site directed mutagenesis and molecular modeling, we estimated the important correlation among motif 1.
In this study, we proposed a catalytic mechanism of Phe SMUG2 on fC and caC through extensive mutational, enzyme kinetics and molecular modeling approaches. A histidine residue in motif 2 could interact with fC and caC, activate the departure of iii
substrates and stabilize the leaving group. We expressed the two zebrafish TDG enzymes and demonstrated their catalytic difference on U, hmU, fC and caC. Mutational analyses and enzyme kinetics suggested the significance of a histidine residue in motif 1 of zTDG2 and we hypothesize this histidine as the specificity determinant in zTDG2. We also compared the predicted structure of zTDG1 and zTDG2 that were predicted by AlphaFold2 and the results indicated the remarkable difference of a certain segment surrounding the histidine residue. The subsequent investigation on this segment in Sfo TDG vindicate the correlation within this segment by series of mutational analyses and catalytic activity analyses. In summary, these functional and structural analyses expand our knowledge about substrate specificity and catalytic mechanism of UDG superfamily which underscores the functional diversity in this superfamily.
Chang, Chenyan, "New DNA Repair and Demethylation Functions in Uracil DNA Glycosylase Superfamily" (2023). All Dissertations. 3313.