Smiley, Adam2024-08-222024-08-222024-06https://hdl.handle.net/11299/265171University of Minnesota Ph.D. dissertation. June 2024. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Wendy Gordon. 1 computer file (PDF); vii, 150 pages.The HUH-endonuclease superfamily is a widespread and diverse group of enzymes that interact with single-stranded DNA (ssDNA) in a sequence-specific manner to mediate a range of biological processes, including rolling circle replication, conjugative plasmid transfer, and DNA transposition. Despite their functional diversity, these enzymes are unified by two universal motifs: a metal-coordinating motif consisting of a pair of histidine (H) separated by a large hydrophobic residue (U) and a catalytic tyrosine, known as the Y motif. All members of the HUH-endonuclease superfamily mediate their functions through the recognition, cleavage, and ligation of ssDNA. Interestingly, their shared mechanism of ssDNA cleavage yields a covalent intermediate in which the Y motif forms an adduct with the newly exposed 5′ end of the enzyme’s cleaved substrate via a phosphotyrosine linkage. Though these covalent protein-ssDNA linkages are transient products in endogenous contexts, bioconjugates formed by reacting recombinant HUH-endonucleases with ssDNA substrates containing their specific cleavage motif are long-lived and non-labile. Recently, these enzymes have been co-opted and developed into ‘HUH-tags’, a versatile bioconjugation platform that mediates simple sequence-directed bioconjugation of proteins of interest to ssDNA substrates of interest. This dissertation focuses on further characterization of HUH-endonucleases –specifically replication initiating HUH-endonucleases (reps)—across their different functions to gain insights that could enhance their performance as HUH-tags for bioconjugation applications and describes efforts to further develop the HUH-tag platform and expand its capabilities, applicability, and versatility. The work presented here includes investigations into the mechanisms of rep function during rolling circle replication, the development of a computational tool for optimizing substrate design in HUH-tag applications, protein engineering efforts to expand HUH-tags beyond ssDNA substrates, and speculation on how to improve this bioconjugation platform moving forward.enThesis or Dissertation