Computational investigation of enzyme-facilitated cleavage of the phosphodiester bond in nucleic acids

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Kaur, Rajwinder
University of Lethbridge. Faculty of Arts and Science
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Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry
The phosphodiester backbone in nucleic acids is remarkably resistant to degradation. Although high stability is essential for storage of genetic information and proper cell function, some circumstances necessitate the cleavage of the nucleic acid backbone. For example, breaking the DNA backbone is critical to repair damage and maintain genetic integrity, while RNA cleavage is necessary for quality control during protein synthesis. Nucleases are enzymes that facilitate the challenging phosphodiester bond cleavage by accelerating the uncatalyzed reaction. Many nucleases utilize metals to enhance catalysis. Despite several experimental studies on enzymes that cleave phosphodiester bonds in nature, the mechanism for bond cleavage used by many enzymes, including the role and/or number of metal ions involved, is still unclear. Computer modeling is a powerful tool to investigate enzyme-catalyzed reaction mechanisms and discern the roles of the metal/s and amino acids involved in the reaction. This thesis uses computational techniques (i.e., quantum mechanics calculations, molecular dynamics simulations, and quantum mechanics–molecular mechanics calculations) to gain an atomic-level understanding of the phosphodiester bond cleavage reaction catalyzed by nucleases, specifically focusing on APE1, I-PpoI, and EndoV. These enzymes are particularly interesting since they either invoke a single metal ion for catalysis, which conflicts with the two-metal mediated mechanism generally proposed for most nucleases, or the metal-dependence is unknown. The mechanistic details uncovered by this thesis will open the door for new and improved applications of these enzymes in the fields of disease diagnostics, genetic engineering, and therapeutics.
Computational , Enzyme , Nucleic acids , Phosphodiester bond , Bond cleavage , Single-metal , Two-metal