DNA Supercoil Unwinding by Variola Virus Type IB Topoisomerase is Accelerated by Superhelical Density and Hindered by Enzyme-DNA Interactions

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dc.contributor.advisorStivers, James T.
dc.contributor.committeeMemberBerger, James M.
dc.contributor.committeeMemberMcMacken, Roger
dc.creatorAnderson, Breeana G.
dc.date.accessioned2016-12-15T07:22:25Z
dc.date.available2016-12-15T07:22:25Z
dc.date.created2015-08
dc.date.issued2015-04-15
dc.date.submittedAugust 2015
dc.date.updated2016-12-15T07:22:25Z
dc.description.abstractMaintenance of negative supercoils in genomic DNA is essential for diverse biological processes performed by enzymes and DNA binding proteins. Type IB topoisomerases play a key role in removing positive and negative supercoils that would otherwise accumulate at replication and transcription forks. These enzymes use a tyrosine nucleophile to cause a break in the phosphodiester DNA backbone and provide a swivel point to unwind supercoils while remaining covalently attached to the DNA via a reversible phosphotyrosine covalent linkage. An interesting mechanistic question is how topoisomerase binding, cleavage, and supercoil unwinding are regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The variola virus type IB topoisomerase (vTopo) has unique high specificity for the target DNA sequence of 5’-CCCTT-3’ and was used to execute mechanistic studies about topoisomerase function that would be challenging to perform using other type IB topoisomerases. In these studies, we have designed and synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site, providing the first highly defined substrates for exploring the effects of superhelical density on DNA binding, reversible strand cleavage, and unwinding by type IB topoisomerases. We observe that DNA binding, cleavage, and religation are independent of superhelical density. In contrast, minicircles with low superhelical densities were found to relax more slowly than highly supercoiled minicircles, suggesting that the level of torque present in the supercoiled DNA dictates how well the rotating DNA end can be captured by the enzyme to reseal the DNA backbone. This was tested with a charge reversal K271E vTopo mutant where the Lys residue is known to interact with the rotating DNA strand. This mutant enzyme unwinds more supercoils per cleavage event, suggesting that enzyme interactions with the rotating DNA segment are important in determining the efficiency of supercoil unwinding. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to play a role in regulating the steady-state superhelical density of DNA domains in the cell.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/39509
dc.languageen
dc.publisherJohns Hopkins University
dc.publisher.countryUSA
dc.subjecttopoisomerase
dc.subjectDNA supercoiling
dc.subjectenzyme kinetics
dc.subjectDNA minicircles
dc.titleDNA Supercoil Unwinding by Variola Virus Type IB Topoisomerase is Accelerated by Superhelical Density and Hindered by Enzyme-DNA Interactions
dc.typeThesis
dc.type.materialtext
thesis.degree.departmentChemical Biology
thesis.degree.disciplineBiochemistry
thesis.degree.grantorJohns Hopkins University
thesis.degree.grantorKrieger School of Arts and Sciences
thesis.degree.levelDoctoral
thesis.degree.namePh.D.
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