DNA is nicked by Fe (2+)-mediated Fenton reactions with 0.5mM H2O2 by oxidants which are sensitive to higher concentrations of H2O2 and ethanol. Moreover, with 0.5mM H2O2 preferential cleavages occur very strongly at dT residues in the sequence RTGR, as well as at (Y)ATTY and YTTA, and at dC in a subset of RCR in addition to some cleavage at RGGG. At 50mM H2O2, on the other hand, DNA nicking is mediated by ethanol-resistant oxidants and preferential cleavages occur only at the nucleoside 5' to each of the dG moieties in the sequence RGGG. We hypothesized that these nicking preferences are brought about by sequence-specific preferences for Fe(2+) binding to DNA and consequent differences in chemical reactivity. Recent unpublished physical studies suggest a novel solution structure of the RTGR motif in duplex DNA, even in the absence of iron, and we propose to complete the structural characterization of this sequence in a duplex oligonucleotide in the presence and in the absence of Fe(2)+ by NMR, fluorescence, time-resolved fluorescence spectroscopy, and X-ray diffraction. Similar structural studies will also be undertaken for the RGGG motif. The RTGR motif, often in tandem- or inverted repeats, is a required region of many promoters that respond to oxidative- or iron stress, and we propose to test whether the unique binding of iron to this sequence is exploited for this regulation. NADH can drive the iron-mediated Fenton reaction to elicit DNA damage, but not NADPH which inhibits that reaction. We propose to complete the test of our hypothesis that cells diminish their NADH pools and increase the ratio of NADPH to NADH pools as a protective response to the presence of reactive oxygen species by determining the first accurate in vivo levels of the pyridine nucleotides before and after H2O2 exposure. These studies will provide important information about metal:DNA interactions, DNA damage by reactive oxygen species generated by Fenton reactions, and cellular responses to iron and oxygen stresses. They will provide basic information about the dynamics of local DNA structures beyond the static B DNA structure. Finally, they ought to help to clarify the basis of degenerative and other diseased states brought about by reactive oxygen species and to suggest regimes to treat, delay or prevent the onset of such conditions.
