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Date of Award
Doctor of Philosophy (PhD)
The cis-syn cyclobutane pyrimdine dimer (CPD) is the major photoproduct resulting from UV irradiation of duplex DNA that results in C to T mutations found in human skin. CPDs with the anti stereochemistry were recently discovered to be formed in human telomeric DNA that adopts a quadruplex structure in vitro and may also play a role in the effects of sunlight in vivo. In this thesis, the effect of telomeric DNA structure on the formation of the anti-CPDs is investigated, as well as the effect of sequence context on cis-syn CPD formation which could help explain the origin of DNA mutation hot spots and cold spots.
Originally it was hypothesized that anti CPDs formed from G-quadruplex structures, but I now show that anti CPDs can also form in human telomeric DNA sequences when complexed with lithium ions that are known to disfavor G-quadruplex formation. I also show that anti CPDs can also form in the presence of potassium ion when selected guanine bases are changed to inosine to interfere with G-quartet formation. Most significantly weI show that anti-CPDs form in sequences containing A’s in place of G’s that cannot form Hoogsteen hairpins, but can form reverse Hoogsteen hairpins. These results suggest that reverse Hoogsteen hairpins may play a hitherto unrecognized role in the biology and photoreactivity of DNA in telomeres, and possibly in other purine-rich sequences found in regulatory regions.
To study sequence context effects, we designed 129-mer sequences containing all 64 possible NPyPyN tetrads (where PyPy is CC, CT, TC, or TT, and N is A, C, G or T), and used a T4 endonuclease gel electrophoresis assay to determine the relative yields of photoproduct formation at the different sites. The results show that CPD yields for different tetrads varied by as much as an order of magnitude. The yield of CPDs under UVC irradiation at a given site decrease in the order TT > TC > CT > CC, whereas the yield of CPDs under UVB irradiation decreased in the order TT > TC > CC > CT. The yield of CPD formation was lowest with a 5’-G and highest with a 5’-T, whereas the yield was lowest with either a 3’-C, G or T, and highest with a 3’-flanking A. We also studied the sequence context effect on photosensitized CPD formation in the presence of acetone and norfloxacin. The results not only show that the efficiency of photosensitized CPD formation depends on the flanking bases, but also indicates that the efficiency depends on the structure and properties of the photosensitizer. These results suggest that the photosensitizers may result in unique mutation spectra that can be used to identify endogenous photosensitizers such as those implicated in chemi-excitation pathways in melanoma induction.
Chair and Committee
John-Stephen A. Taylor
Mikhail Berezin, Robert Blankenship, Michael L. Gross, Timothy Wencewicz,
Lu, Chen, "Effect of DNA Sequence Context, DNA structure, and Excitation Method on Cyclobutane Pyrimidine Dimer Formation" (2018). Arts & Sciences Electronic Theses and Dissertations. 1559.
Available for download on Wednesday, April 24, 2019