Date Approved

7-2019

Document Type

Dissertation

Degree Name

PhD in Cell & Molecular Biology

Department

Molecular Biology

College

Graduate School of Biomedical Sciences

First Advisor

Salvatore Caradonna, PhD

Committee Member 1

Eric Moss, PhD

Committee Member 2

Michael Anikin, PhD

Committee Member 3

Susan Muller-Weeks, PhD

Committee Member 4

Jennifer Fischer, PhD

Subject(s)

dUTP pyrophosphatase, Nucleotidyltransferases, Protein Isoforms, Oxidation-Reduction, Humans

Disciplines

Cell Biology | Laboratory and Basic Science Research | Life Sciences | Medicine and Health Sciences | Molecular Biology | Molecular Genetics

Abstract

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.

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