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- Genre: Masters Thesis
Description
Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3′ and 5′-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe·BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported.
ContributorsSegerman, Zachary (Author) / Hecht, Sidney M. (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly behavior of a chemo-mechanical protein were explored to better understand their operation. In the first study a photoconvertible fluorescent protein (pcFP) was examined which undergoes a photochemical transformation upon irradiation with blue light resulting in an emission wavelength change from green to red. Photo-transformable proteins have been used in high resolution, subcellular biological imaging techniques, and desires to engineer them have prompted investigations into the mechanism of catalysis in pcFPs. Here, a Kinetic Isotope Effect was measured to determine the rate-limiting step of green-to-red photoconversion in the reconstructed Least Evolved Ancestor (LEA) protein. The results provide insight on the process of photoconversion and evidence for the formation of a long-lived intermediate. The second study presented here focuses on the AAA+ protein Rubisco activase (Rca), which plays a critical role in the removal of inhibitors from the carbon-dioxide fixing enzyme Rubisco. Efforts to engineer Rubisco and Rca can be guided by a deeper understanding of their structure and interactions. The structure of higher plant Rca from spinach, and its interactions with its cognate Rubisco, were investigated through negative-stain electron microscopy (EM) and cryo-EM experiments. Multiple types of higher-order oligomers of plant Rca were imaged which have never been structurally characterized, and the AAA+ core of plant Rca was shown to bind Rubisco side-on, similar to bacterial Rca’s. Higher resolution structures of these aggregates and complexes are needed to make definitive observations on protein-protein interactions. However, the results presented here provide evidence for the formation of regulatory structures and an experimental foundation for future exploration of plant Rca through cryo-EM.
ContributorsBreen, Isabella (Author) / Wachter, Rebekka (Thesis advisor) / Chiu, Po-Lin (Thesis advisor) / Levitus, Marcia (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2020