Investigation of DNAzymes catalyzing thymine dimer photoreactivation and phosphodiester cleavage using a new method of iodine-mediated phosphorothioate cross-linking

Resource type
Thesis type
((Thesis)/(Dissertation)) Ph.D.
Date created
2012-05-25
Authors/Contributors
Abstract
In recent years, many artificial and naturally occurring catalytic nucleic acids molecules have been discovered. These enzymes, composed purely of RNA or DNA, are referred to as ribozymes or DNAzymes. With an ever-increasing repertoire of chemical reactions they are able to catalyze, researchers have attempted to discover the mechanisms governing catalysis by identifying the functional groups involved in catalysis. This thesis introduces a novel Iodine-mediated phosphorothioate cross-linking (IMPC) method, which enables structure mapping around the phosphodiester backbone of DNA. Two DNAzymes that I have investigated using this method are the 8-17 RNA-cleaving enzyme and the UV1C cyclobutane thymine dimer-repairing enzyme. The UV1C DNAzyme adopts a G-quadruplex structure that is responsible for enhanced, in comparison to non-G-quadruplex forming DNA, absorption of light energy of >300 nm wavelength to photo-reactivate cyclobutane thymine dimers in a DNA substrate. IMPC results suggest that the topology of this G-quadruplex is an all-parallel propeller orientation. UV1C was originally in vitro selected for repair of a substrate lacking an intra-dimer phosphodiester phosphate. I show that UV1C can catalyze repair of thymine dimer mutations in a “natural” intact single stranded DNA substrate at wavelengths greater than 300 nm. Additionally, evidence is produced indicating that UV1C shifts the photostationary state of thymine dimer formation to favour monomer formation in comparison to single stranded and double stranded DNA at 280 nm wavelength. While other biochemical cross-linking methods explore base-to-base contacts, IMPC identifies base-to-backbone contacts. This provides an advantage at identifying phosphodiester cleaving ribozyme and DNAzyme nucleobases directly involved in phosphodiester cleavage at the enzyme’s active site. Using this approach, not only is the 8-17’s catalytic core base contacts identified, but also a chirally resolved phosphorothioate at the cleavage site provides a stereochemical glimpse of a key cytosine (C13) base. Another important cytosine (C3) is also identified, for the first time. On the basis of the C3’s proximity to the cleavage site and the impact of its mutation on the DNAzyme’s catalytic rate, it is functionally implicated to perform an acid-base role in catalysis.
Document
Identifier
etd7283
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The author granted permission for the file to be printed, but not for the text to be copied and pasted.
Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Sen, Dipankar
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etd7283_GSekhon.pdf 15.25 MB