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Elucidating the Basis for Substrate-Dependent Control of [4Fe4S] Redox Properties in Human DNA Primase

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dc.contributorFrantom, Patrick A.
dc.contributorVincent, John B.
dc.contributorPapish, Elizabeth T.
dc.contributorBowman, Michael K.
dc.contributorSummers, Ryan M.
dc.contributor.advisorThompson, Matthew K.
dc.contributor.authorPetersen, Courtney
dc.date.accessioned2024-09-17T16:18:49Z
dc.date.available2024-09-17T16:18:49Z
dc.date.issued2024
dc.descriptionElectronic Thesis or Dissertationen_US
dc.description.abstractIn eukaryotes, the fundamental tasks of DNA replication, processing, and repair are carried out via the coordinated action of several functionally diverse multiprotein machines. An increasing number of these proteins have been found to coordinate essential high potential [4Fe4S] clusters, though the exact roles these cofactors play in many of these systems remains poorly defined. Still, there is an innate need to understand the roles [4Fe4S] clusters play in these life-sustaining processes, as defects in [4Fe4S]-coordinating domains have been linked to cancer and other diseases of genomic instability. To date, most of the information available regarding the functional roles [4Fe4S] clusters play in eukaryotic DNA replication stem from studies involving DNA primase. During replication, DNA primase synthesizes short ribonucleotide primers that, after further modification, are used for processive DNA replication by the canonical polymerases δ and ϵ. Electrochemical studies conducted with human DNA primase have demonstrated DNA binding in the presence of ribonucleotide triphosphates (rNTPs) activates the [4Fe4S] cluster towards reversible redox activity. This observation implies rNTP binding 25 Å away allosterically modulates the redox properties of the [4Fe4S] cluster, however, the structural basis for this effect has not yet been explored. The purpose of this dissertation is to investigate the root cause of this behavior using a combination of spectroscopic approaches. Chapter 2 describes the results of chemical titration experiments in which oxidation of the [4Fe4S] cluster in p58C was monitored by Electron Paramagnetic Resonance spectroscopy. Chapter 3 then discusses the results of resonance Raman, Nuclear Resonance Vibrational Spectroscopy, and Fe K-edge Extended X-ray Absorption Fine Structure experiments focused on investigating the influence of substrate binding on cluster structure. Taken together, the results presented herein suggest the substrate-dependent redox behavior of the [4Fe4S] cluster is not exclusively caused by changes to cluster geometry and that other factors may be present that affect the immediate environment surrounding the cluster. Given the 25 Å distance between the [4Fe4S] and DNA binding sites, the work presented in this dissertation implicates the 5'-triphosphate of the initiating rNTP and several protein residues in an allosteric mechanism responsible for the communication of redox state information.en_US
dc.format.mediumelectronic
dc.format.mimetypeapplication/pdf
dc.identifier.other1089556
dc.identifier.urihttps://ir.ua.edu/handle/123456789/14461
dc.languageEnglish
dc.language.isoen_US
dc.publisherUniversity of Alabama Libraries
dc.relation.hasversionborn digital
dc.relation.ispartofThe University of Alabama Electronic Theses and Dissertations
dc.relation.ispartofThe University of Alabama Libraries Digital Collections
dc.rightsAll rights reserved by the author unless otherwise indicated.en_US
dc.subjectallostery
dc.subjectDNA primase
dc.subjectelectron paramagnetic resonance spectroscopy
dc.subjectvibrational spectroscopy
dc.subject[4Fe4S] cluster
dc.titleElucidating the Basis for Substrate-Dependent Control of [4Fe4S] Redox Properties in Human DNA Primaseen_US
dc.typethesis
dc.typetext
etdms.degree.departmentUniversity of Alabama. Department of Chemistry and Biochemistry
etdms.degree.disciplineChemistry
etdms.degree.grantorThe University of Alabama
etdms.degree.leveldoctoral
etdms.degree.namePh.D.

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