Identifying the Molecular Mechanisms of Thymine DNA Glycosylase (TDG) Substrate Specificity Item Type dissertation Authors Dow, Blaine Jacob Publication Date 2021 Abstract Thymine DNA glycosylase (TDG) helps maintain genomic integrity by removing thymine from G·T mispairs arising via deamination of 5-methylcytosine (mC). TDG employs strict regulation for both the opposing guanine, as well as the base downstream of the ... Keywords Substrate Specificity; Thymine DNA Glycosylase--chemistry Download date 25/09/2021 04:04:58 Link to Item http://hdl.handle.net/10713/15771 CURRICULUM VITAE BLAINE “JAKE” DOW Email: [email protected] (619)270-6122 EDUCATION Institution Degree Year Awarded Major Univ of Maryland Baltimore Ph.D. May 2021 Biochemistry Baltimore, MD Advisor: Dr. Alex Drohat Univ of California--San Diego B.S. 2011 Human Biology La Jolla, CA PROFESSIONAL EXPERIENCE Graduate Research Assistant University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology Advisor: Dr. Alex Drohat (February 2016-present) ▪ Studied substrate specificity for thymine DNA glycosylase (TDG) by analyzing structure-function relationships, employing a combination of enzyme kinetics, NMR, ITC, and x-ray crystallography. ▪ Developed novel method using 19F NMR to quantify base flipping into the enzyme active site ▪ Identified key amino acid residues via mutational analysis ▪ Identified difference in specificity for key TDG physiological substrates Lab rotations ▪ Nov 2015- Feb 2016 w/ Dr. Eric Sundberg: studied the interaction between H. pylori adhesion protein HopQ and dimer interface of human CEACAM proteins. Performed fluorescence-activated cell sorting (FACS; Biorad); directed evolution via yeast display using CEACAM mutagenic library, HopQ as free ligand; PCR; protein purification via FPLC. ▪ Jul 2015- Nov 2015 w/ Dr. Christopher Plowe: quantified drug resistance mutations in malaria parasites (P. falciparum) collected from humans vs mosquitos. Performed qPCR, pyrosequencing ▪ Mar 2015- Jul 2015 w/ Dr. Martin Schneider: Isolated and cultured individual skeletal muscle fibers from mouse FDB muscle (in foot); transduced these muscle fibers with adenovirus containing GFP-tagged FOXO1 protein (transcription factor), then determined changes in the ratio of FOXO1 in nucleus vs. cytoplasm, via confocal microscopy, upon electrical stimulation. Also produced more adenovirus via HEK 293 cells. ▪ Dec 2014- Mar 2015 w/ Dr. Katia Kontrogianni: studied binding of myosin- binding protein C to both myosin and actin via pull-down/Western blot, using various mutations mimicking phosphorylation at key amino acid residues. US Air Force (Jan 2001- Jan 2007) ▪ Electronic Warfare Technician: Supervised swing shift crew of 6 people, maintained aircraft defensive systems, led squadron fitness program, instructed flare loading/unloading classes. AWARDS & HONORS ▪ Summa cum laude B.S. in Human Biology UCSD ▪ Poster Award Winner, 2020 Baltimore Area Repair Symposium ▪ Poster Award Winner, 2018 Baltimore Area Repair Symposium ▪ Honor graduate, US Air Force basic training, 2001 ▪ Distinguished graduate, US Air Force basic electronics course, 2001 ▪ Distinguished graduate, US Air Force electronic warfare technical school, 2001 PUBLICATIONS • Dow, B. J., Drohat, A. C. Identifying the molecular mechanisms of opposing base specificity during removal of thymine by TDG. Manuscript in preparation. • DeNizio, J. E., Dow, B. J., Serrano, J. C., Ghanty, U., Drohat, A. C., and Kohli, R. M. (2021) TET-TDG active DNA demethylation at CpG and non-CpG sites. J Mol Biol. 433(8), 166877. • Dow, B. J., Malik, S. S., and Drohat, A. C. (2019) Defining the Role of Nucleotide Flipping in Enzyme Specificity Using 19F NMR. J Am Chem Soc 141, 4952-4962. • Tarantino, M. E., Dow, B. J., Drohat, A. C., and Delaney, S. (2018) Nucleosomes and the three glycosylases: High, medium, and low levels of excision by the uracil DNA glycosylase superfamily. DNA Repair (Amst) 72, 56-63. • Bonsor, D. A., Zhao, Q., Schmidinger, B., Weiss, E., Wang, J., Deredge, D., Beadenkopf, R., Dow, B., Fischer, W., Beckett, D., Wintrode, P. L., Haas, R., and Sundberg, E. J. (2018) The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA. The EMBO journal 37. PRESENTATIONS ▪ Dow, J., Drohat, A.C. (2020) Differing context specificity for thymine DNA glycosylase (TDG) substrates. Baltimore Area Repair Symposium (poster). ▪ Dow, J., Malik, S., Drohat, A.C. (2019) Studying Thymine DNA Glycosylase (TDG) CpG Specificity Using 19F NMR. EMGS Special Symposium. Genome Maintenance Systems in Cancer Etiology and Therapy: A Tribute to Paul Modrich. Washington DC (oral/poster). ▪ Dow, J., Drohat, A.C. (2019) Differing context specificity for thymine DNA glycosylase (TDG) substrates. University of Maryland, Baltimore Graduate Student Research Conference (poster). ▪ Dow, J., Drohat, A.C. (2018) Differing context specificity for thymine DNA glycosylase (TDG) substrates. Baltimore Area Repair Symposium (poster). ▪ Dow, J., Drohat, A.C. (2018) Differing context specificity for thymine DNA glycosylase (TDG) substrates. University of Maryland, Baltimore Graduate Student Research Conference (poster). ▪ Dow, J., Drohat, A.C. (2017) Investigating the CpG context specificity for excision of 5-formylcytosine and 5-carboxylcytosine by Thymine DNA Glycosylase. University of Maryland, Baltimore Graduate Student Research Conference (poster). TECHNICAL EXPERIENCE AND EXPERTISE ▪ Protein purification (via FPLC, AKTA pure) ▪ SDS PAGE/Western blot ▪ DNA purification (via HPLC, Varian) ▪ Sample analysis (via UHPLC, Thermo Fisher Vanquish/Chromeleon) ▪ Enzyme kinetics: single-turnover, analyzed by gel and chromatographic methods ▪ Sample preparation and screening of crystallization conditions ▪ cloning and mutagenesis ▪ Isothermal titration calorimetry (ITC) ▪ 1D 19F NMR Abstract Title of Dissertation: Identifying the Molecular Mechanisms of Thymine DNA Glycosylase (TDG) Substrate Specificity Blaine J. Dow, Doctor of Philosophy, 2021 Dissertation Directed by: Dr. Alexander Drohat, Ph.D., Associate Professor, Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland Baltimore Thymine DNA glycosylase (TDG) helps maintain genomic integrity by removing thymine from G·T mispairs arising via deamination of 5-methylcytosine (mC). TDG employs strict regulation for both the opposing guanine, as well as the base downstream of the target thymine, in order to limit removal of thymine from canonical A·T pairs, as erroneous removal of thymine from A·T pairs is mutagenic and cytotoxic. TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidation products of mC generated by ten-eleven translocation (TET) enzymes during active DNA demethylation. Remarkably, using single-turnover kinetics reactions to determine the maximum rate of substrate removal, kmax, we find that TDG activity for fC and caC shows little dependence on the opposing base or the downstream base, revealing a major difference in specificity for excision of fC and caC relative to T. Using a novel 19F NMR approach to determine the flipping equilibrium for thymine into the TDG active site, we establish that specificity during thymine excision manifests largely by modulating the stability for thymine flipping in the active site. Structure-function analysis employing a variety of opposing bases reveals that both the thermodynamic stability (ΔH) of A·T pairs, as well as direct contacts between TDG and the opposing base, contribute to opposing base specificity. The differences in specificity observed for thymine versus fC/caC are likely explained by interactions between these substrates and the TDG active site. Structural information obtained from x-ray crystallography, combined with TDG mutational studies, identified several TDG active site residues that form stabilizing interactions with fC and caC, helping to both stabilize base flipping into the active site, as well as enhance the chemical steps of base excision. Conversely, two conserved residues in TDG, A145 and H151, limit stability of thymine in the active site, and destabilize thymine as a leaving group. As a result, additional contacts between both the opposing guanine, as well as the guanine downstream of the target thymine, appear necessary to orient thymine in a manner which produces stable, productive flipping into the active site. Identifying the Molecular Mechanisms of Thymine DNA Glycosylase (TDG) Substrate Specificity by Blaine J. Dow Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2021 Acknowledgements First and foremost, I would like to thank Dr. Alex Drohat, for being such a great mentor. He gave me enough independence to allow me to grow as a researcher, but was always available when I needed his guidance or support. I would also like to thank Dr. Swarna Pidugu, for her willingness to share her expertise in protein purification, and countless other lab hacks/techniques. And thank you to Dr. A-Lien Lu-Chang, for her valuable insights and constructive criticism during lab meetings, which helped me learn to present my findings. iii Table of Contents Chapter 1: Introduction ....................................................................................................... 1 1.1-- Overview ...............................................................................................................