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Date of Award
Doctor of Philosophy (PhD)
G-quadruplexes are non-canonical DNA structures that form in G-rich sequences and are thought to have biological significance due to the presence of putative G-quadruplex forming sequences in promoter regions and in telomeres. G-quadruplex structures can fold into a variety of stable conformations that are influenced by sequence, loop length and coordinating cation. Our group had previously discovered that irradiation of a short fragment of telomeric DNA, d[AAAGGG(TTAGGG)3AA] (Tel26), with UVB light results in the formation of a significant amount of an anti cyclobutane pyrimidine dimer (CPD) between two separated T’s. The most likely G-quadruplex conformation that would facilitate a photocrosslinking reaction between thymines in loops 1 and 3 would involve a basket-type conformation. However, the anti CPD was only observed when Tel26 was in the presence of potassium, but not when sodium ions were present. These results were contrary to our hypothesis since Tel26 in sodium has been characterized to fold into a stable basket structure with two proximal loops, while Tel26 in potassium folds into a Hybrid (3+1) structure with one exterior loop.
These findings led us to explore possible intermediate conformations of the Tel26 equilibrium that would facilitate the loop photocrosslinking that leads to anti CPD formation. We investigated the formation of the anti CPD with a series of substitutions and insertions to the Tel26 sequence in order to stabilize the structurally related two-tetrad basket G-quadruplex conformation, Form 3. We show evidence that key positions in the stability of the Form 3 conformation are important for the formation of anti CPD in Tel26. We also investigated the formation of anti CPD in the presence of various coordinating cations and in long sequences of telomeric DNA. We then extended our study of G-quadruplex loop photocrosslinking to include promoter region G-quadruplex sequences. We identified and characterized several promoter region Gquadruplex sequences by circular dichroism, melting temperature analysis and chemical footprinting techniques. Irradiation of the promoter region G-quadruplexes with UVB light led to the identification of several nonadjacent photoproducts that resulted from loop photocrosslinking.
We also report the discovery of an anti thymine-cytosine cyclobutane pyrimidine dimer. Future work in the area of G-quadruplex photochemistry is needed to better understand the conditions that facilitate the formation of anti cyclobutane pyrimidine dimers and whether these photoproducts are able to form in vivo.
Chair and Committee
John-Stephen A. Taylor
Robert E. Blankenship, Michael L. Gross, Joshua A. Maurer, Sheila A. Stewart, Hani S. Zaher
Smith, Jillian Eleanore, "Anti Thymine Photodimer Formation in Human Telomeric DNA and Putative G-Quadruplex Forming Sequences in Promoter Regions" (2013). Arts & Sciences Electronic Theses and Dissertations. 1030.