The Role of Protein-Nucleic Acid Interactions in the Function and Fidelity of DNA Polymerase β and CRISPR Cas9 Monitored by NMR
出版項
2019
說明
1 online resource (148 pages)
文字
text
無媒介
computer
成冊
online resource
附註
Source: Dissertations Abstracts International, Volume: 81-03, Section: B
Advisor: Loria, J. Patrick
Thesis (Ph.D.)--Yale University, 2019
Includes bibliographical references
Protein-nucleic acid interactions are one of the most important processes required for life on earth. These interactions make up the core of the central dogma of molecular biology first published by Francis Crick in 1958. For over six decades, the interactions of proteins with DNA and RNA have been heavily studied in order to better understand how the proteome interacts with the genome and nucleic acid polymers. The complexity of these interactions is best highlighted by the thousands of proteins that interact with genomic DNA and various RNAs. These protein-nucleic acid interactions are required for the integrity of the genome and the stability of the processes required by the central dogma. The integrity of genomic DNA requires the fidelity of the DNA polymerases that copy and repair the millions of DNA base pairs per cell. One polymerase that repair DNA in humans is DNA polymerase β. Studies of DNA polymerase β (pol β) have shown that the interactions of pol β with the target DNA are tightly regulated to maximize the fidelity of the enzyme. The wild type enzyme was monitored using NMR spectroscopy in order to study the effects of the four template nucleotides (adenosine, guanosine, thymine, and cytosine) have on the structure and dynamics of the enzyme. The wild type enzyme was shown to bind independently of the nucleotide; however, the steric effects of the template nucleotide and the incoming nucleotide triphosphate show that the mechanism for selecting the appropriate nucleotide is heavily dependent on the steric effects of the base pair. To further these studies, several cancer-associated mutants of pol β have been identified that are still active polymerases but with reduced fidelity. One of these cancer-associated mutants, E288K, has a reduced fidelity only when the templating nucleotide is adenosine. This reduced fidelity allowed for the study of how the mutation of E288K altered the interaction of pol β with the target DNA.The CRISPR Cas9 endonuclease is an important molecular system for genome editing and the future of gene therapy. NMR studies of the entire Cas9 enzyme are hindered by the large size of the enzyme-RNA complex. Therefore, a construct of the HNH endonuclease domain of Cas9 was designed to allow for the assignment and study of part of Cas9. The HNH construct was assigned to 85%, allowing for the characterization of the internal structure and dynamics of the enzyme in solution by NMR. The HNH construct is structurally indistinguishable from the HNH domain of the full length Cas9 enzyme by x-ray crystallography. The measurement of dynamics on multiple time scales by NMR adds further evidence to the suggestion that the HNH domain is necessary for the communication between the PAM recognition domain and the second endonuclease domain, RuvC. This communication pathway is of great interest in the intelligent design of Cas9 enzymes for future gene therapy techniques
Electronic reproduction. Ann Arbor, Mich. : ProQuest, 2020
