PhD in Cell & Molecular Biology
Graduate School of Biomedical Sciences
School of Osteopathic Medicine
New Jersey Health Foundation (PC11-1; to MH)
Salvatore Caradonna, PhD
Committee Member 1
Eric Moss, PhD
Committee Member 2
Michael Anikin, PhD
dUTP pyrophosphatase, Nucleotidyltransferases, Protein Isoforms, Oxidation-Reduction, Humans
Cell Biology | Laboratory and Basic Science Research | Life Sciences | Medicine and Health Sciences | Molecular Biology | Molecular Genetics
Deoxyuridine nucleotidyl transferase (dUTPase) is an enzyme found in all organisms that have thymine as a component of DNA. It catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate thus precluding the buildup of dUTP pools as well as providing the substrate, dUMP, for the de novo synthesis of thymidylate. In Homo sapiens, there are four isoforms: mitochondrial (mDut), nuclear (nDut), variant 3 and variant 4. This work is largely focused on nDut. Using structural and MS analyses of recombinant dUTPase constructs, an intermolecular disulfide bridge between cysteine-3 of each nDut monomer was discovered. It was found that these two residues stabilize a dimer configuration that is unique to the nDut isoform. It was also uncovered that there is an intramolecular disulfide linkage between cysteine residues C-78 and C-134, stabilizing the monomeric state of the protein. Both disulfide linkages are essential for nDut’s enzymatic activity and the dimeric form can be augmented by the addition of the oxidizing agent, hydrogen peroxide. It was observed that mDut appears to be a mixture of monomer, dimer, and trimer conformations, as well as higher-order subunit interactions. In contrast, nDut appeared to exist only in monomeric and dimeric forms.
Cysteine-based redox “switches” have recently emerged as a distinct class of post-translational modifications that have been shown to influence subcellular location, turnover rate, and catalytic activity of many enzymes. Taking this work as a whole we propose that nDut possesses a redox switch that regulates nDut’s dUTP- hydrolyzing activity.
Moreover, in recent years dUTPase in multiple organisms has been implicated to be involved in a secondary function outside of hydrolysis of dUTP. With the discovery of the two additional human isoforms, this work has laid the foundation to begin to elucidate their function. This is the first report of a viable mammalian dUTPase knockout cell lines. The work presented here validates this line as a useful tool for the individual analysis to evaluate the contributions of each of the four human dUTPase isoforms.
Rotoli, Shawna Marie, "Characterization of Human dUTPase" (2019). Graduate School of Biomedical Sciences Theses and Dissertations. 37.