Development of LC-MS for the Identification and characterization of non-adjacent DNA Photoproduct Formation in G-quadruplex Forming Sequences
Date of Award
Master of Arts (AM/MA)
Ultraviolet light is well known to induce cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts in duplex DNA, which interfere with DNA replication and transcription. Recently, a new class of DNA photoproducts known as anti cyclobutanepyrimidine dimers have been discovered, which form in G-quadruplex forming sequences in solution. G-quadruplex structures have been proposed to form in human DNA telomeres and certain promoters in vivo but evidence for their existence has been lacking. Since anti-cyclobutante pyrimidine dimers have been shown to form in G-quadruplex forming sequences, their formation in irradiated human cells could be used to confirm the existence of G-quadruplexes in vivo. Methods have been developed for assaying these DNA modifications, but it hasn’t always been easy to confirm their identity or characterize their structures.
The goal of this thesis was to further investigate the formation of anti-thymine dimers in human telomeric sequences under various conditions known to favor or disfavor G quadruplex formation. Central to this goal was the development of a high resolution LC MS/MS method for separating and identifying all the photoproducts. Part one of this thesis, describes the development of a new LC method to better separate the CPD-containing nuclease P1 (NP1) digestion products of DNA and the utilization of the new method to analyze the NP1 products of UV irradiated telomeric DNA isolated with the previous HPLC method. The second part of this thesis focused on directly coupling the LC method to MS/MS mass spectrometry to simultaneously separate and identify the nuclease P1 digestion products of UV irradiated telomeric DNA in Li+. These products were then compared against the products formed in the presence of K+. In the course of these studies new hexameric digestion products corresponding to (pTTA)2 were also identified which could contain two CPDs dimers.
Chair and Committee
Dr. John-Stephen A. Taylor
Dr. Kevin Moeller Dr. Robert E. Blankenship
Posadas, Claudia, "Development of LC-MS for the Identification and characterization of non-adjacent DNA Photoproduct Formation in G-quadruplex Forming Sequences" (2018). Arts & Sciences Electronic Theses and Dissertations. 1291.
Biochemistry Commons, Biotechnology Commons, Molecular Biology Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Organic Chemicals Commons
Permanent URL: https://doi.org/10.7936/K72J6B8C