Date of Award

5-2013

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

Johannes Schelvis

Committee Member

Yvonne Gindt

Committee Member

Mark Whitener

Abstract

Skin cancer is the most prevalent malignancy found in humans, with the diagnosis rate continuing to steadily increase. The primary cause of this disease is overexposure to harmful ultraviolet (UV) radiation from sunlight, which can induce damage to the nitrogenous bases in DNA via dimerization. The most prevalent UV-induced photoproducts in DNA are cyclobutane pyrimidine dimers (CPDs), most commonly between adjacent thymidines.

Organisms have implemented methods by which to repair these CPDs, the primary of which are nucleotide excision repair (NER) and photoreactivation by photolyases. Photolyases are blue-light activated flavoproteins that are more efficient at recognizing and repairing CPDs than the NER enzymes relied on by humans. Thus, understanding the interactions between photolyase and DNA may lead to improved treatments for the harmful effects from UV radiation.

UV-irradiated oligothymidylates are the most commonly used models for studying DNA-bound photolyases, as it is possible to quickly form a high concentration of CPDs. However, these strands present a random distribution of CPDs and other photoproducts can be present within the strands. This research examines potential differences between single and double strand oligothymidine interactions with the neutral radical semiquinone flavin adenine dinucleotide (FADH’) cofactor within the active site of E. coli photolyase. This study also investigates whether photolyase-DNA interactions are satisfactorily modeled by using UV-irradiated oligothymidines.

An oligothymidine decamer, p(dT)10, was irradiated with 254 nm UV light to form an average of ~1.5 CPDs per strand. An oligonucleotide dodecamer, 5'-CGGCATTACGGC-3', was irradiated with 302 nm UV light in the presence of acetophenone, a photosensitizer, and the CPD-containing strands were purified using reverse phase HPLC. Single strand UV-p(dT)10, double strand UV-p(dT*dA)10, and the CPD-oligonucleotide were complexed with E. coli photolyase and analyzed using electronic absorption spectroscopy and resonance Raman spectroscopy, with excitation at 532 nm to enhance the vibrations of the FADH*.

All studied oligonucleotides induced very similar electrochromic shifts within the absorption spectrum of photolyase. The resonance Raman spectrum of photolyase exhibits similar changes in the presence of both the single and double strand UV-p(dT)10, indicating comparable interactions within the active site. The Raman spectrum of the CPD-oligonucleotide-photolyase complex differs significantly from that of the UV-p(dT)10 complex, indicating that oligothymidylates may not serve as good models in photolyase-DNA studies. The flavin cofactor exhibits a dramatically faster rate of reduction in the presence of the CPD-oligonucleotide than the UV-oligothymidylate. However, this may be a side effect of triethylamine from the HPLC.

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