Part I. NMR studies of sequence-specific transition metal binding to biologically important DNA sequences cleaved during the iron-mediated Fenton reaction. Part II. DNA sequence-dependent steady-state and time-resolved fluorescence characteristics of 2-

The object of the research reported in this thesis was to use Nuclear Magnetic Resonance (NMR) spectroscopy to understand the DNA sequence-specificity of cleavage generated by the iron-mediated Fenton reaction by investigating the sequence dependence of transition metal localization within duplex DNA. Nicking of duplex DNA by the bimodal iron-mediated Fenton reaction occurs preferentially at a limited number of sequences which differ for the two kinetic modes, Mode I and Mode II. Of these, the major Mode I sequence purine-T-G-purine (RTGR) and the exclusive Mode II sequence purine-GGG (RGGG) are of particular interest. RTGR is a required element in the upstream regulatory regions of many genes involved in iron- and oxidative stress responses and RGGG is part of the mammalian telomeric repeat, TTAGGG.; In order to study the basis of this preferential nicking, NMR studies of Fe2+ localization were undertaken on RTGR-containing and RGGG-containing duplex oligonucleotides and various related derivative oligonucleotides. One-dimensional and two-dimensional 1H NMR measurements show preferential and reversible Fe2+ association at the consensus site within both duplexes, at a rate that is rapid relative to the chemical shift timescale. Selective paramagnetic NMR line-broadening of the guanine H8 and 7-deazaguanine substitutions for guanine within the consensus sequences suggests that Fe2+ interacts with the guanine N7 moiety.; 2-Aminopurine (2-AP), a fluorescent analog of adenine, has been widely used as a probe for local DNA conformation, since excitation and emission characteristics and fluorescence lifetimes of 2-AP vary in a sequence-dependent manner within DNA. Using steady-state and time-resolved fluorescence techniques, I report that 2-AP appears to be unusually stacked in the internal positions of ATAT and TATA in duplex DNA. The excitation wavelength maxima for 2-AP within these contexts are red-shifted, indicating a lower energy, stacked conformation for the fluorophore. Furthermore, in these contexts, 2-AP fluorescence is resistant to acrylamide-dependent collisional quenching, suggesting that the fluorophore is protected by its stacked position within the duplex. This conclusion is further reinforced by the presence of a peak at 275 nm in the excitation spectra that is indicative of direct excitation energy transfer from nearby non-fluorescent DNA bases. (Abstract shortened by UMI.)...