Date of Award

5-2014

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Yvonne M. Gindt

Committee Member

Johannes P. M. Schelvis

Committee Member

James Dyer

Committee Member

Marc L. Kasner

Abstract

Vibrio cholerae cryptochrome 1 (VcCryl), a DNA repair enzyme, uses a lightdriven electron transfer to repair UV damaged single strand DNA but not double strand DNA. VcCryl has an active site FAD which is required for substrate binding and repair. Size exclusion chromatography (SEC) was used to observe changes in oligomer structure when changes in the oxidation state of the FAD cofactor were made. SEC studies found the oxidized state exists as a dimer with an observed mass of 163 kDa in solution, in dynamic equilibrium with a 67 kDa fragment. The reduced state exists as a monomer in solution, while the semiquinone state exists as a 51 kDa structure. SEC was also used to observe the influence of substrate to oligomer structure of VcCryl. It was found that the previously mentioned dimer found in the oxidized state was lost when substrate was present. The reduced and semiquinone states appeared as monomers in the presence of substrate.

Isothermal titration calorimetry was used to study the temperature dependence of DNA binding to VcCryl. The thermodynamics important in the VcCryl substrate binding were examined and compared to E. coli photolyase, a related DNA repair enzyme. Similar trends in the thermodynamic parameters were observed between VcCryl and E. coli photolyase.

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