Kingston, RobertWalker, SuzanneLevine, Zebulon Gehrett2019-12-122019-052019-05-152019Levine, Zebulon Gehrett. 2019. Strategies to dissect O-GlcNAc Transferase biochemistry in vitro and in cells. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029809In this dissertation, I develop tools to allow us to understand how O-GlcNAc Transferase (OGT) recognizes its substrates and to separate OGT's catalytic functions in a cellular context. In the introduction, I introduce OGT's multiple catalytic activities and examine why they are necessary for mammalian cell survival. In the second chapter, I use targeted and high-throughput methods to determine the role of OGT's N-terminal tetratricopeptide repeat (TPR) domain in substrate selection. I demonstrate that the majority of substrate are bound in the lumen of this superhelical domain, with conserved Asn binding the peptide backbone and key Asp residues playing a selectivity-determining role. In the third chapter, I develop a system for replacing OGT with enzyme variants in living cells; this system allows me to conclude that O-GlcNAc transfer to Ser/Thr, not HCF-1 cleavage, is essential for cell viability. Together, these chapters present methods that can be applied for both further biochemical studies of OGT substrate selection as well as future OGT structure-function studies in cells. Last, I present an appendix on by-residue conservation of OGT that may guide such future studies.application/pdfenO-GlcNAc, enzymology, biochemistry, rapid degradation, substrate recognition, OGTThesis or Dissertation2019-12-120000-0002-0830-9032