The long-term objective of this proposed research program is to (i) measure the types, yields and spatial distribution of the biologically relevant DNA lesions produced by the direct effect of ionizing radiation, (ii) determine the chemical mechanisms by which these DNA lesions are formed, and (iii) evaluate how molecules such as histones and other DNA binding proteins modulate the yield and distribution of these DNA lesions. Achievement of these objectives should make it possible to predict the composition and spatial distribution of the DNA lesions within biologically important clusters. This predictive capability is central to making risk/benefit decisions at both low dose rates and low doses of radiation and to constructing biologically relevant models of clustered DNA damage to be used by DNA enzymologist, biochemists and biophysicists in their DNA repair/misrepair studies.
The specific aims are: 1) to determine the yields of specific sugar and base damage end-products produced by the direct effect as a function of radiation dose, and elucidate the reaction mechanisms giving rise to them, 2) to determine the yield and composition of DNA damage within clusters produced by the direct effect, and 3) to determine how histones and other protein DNA binding complexes modify the yield and composition of clustered DNA damage produced by the direct effect. Damage to DNA by the direct effect occurs via two routes. One is by direct ionization of the DNA. The other is by ionization of that portion of the solvent shell that is tightly bound to the DNA and rapid transfer of that damage to the DNA. Because of these properties, information on the direct effect is optimized by studying DNA in the solid state. The DNA samples, for this proposed research program, will be prepared in the form of crystals and films. Using crystals of known structure, we maximize our knowledge of relevant parameters: base sequence, conformation, hydration state, counter ions, packing, and purity. Using films we are able to vary parameters such as base sequence and degree of hydration. Unstable free radical intermediates will be studied by electron paramagnetic resonance (EPR) spectroscopy. Stable end products will be quantified by high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS). Stable end products will be analyzed using the same samples as those studied by EPR. Key elements in our experimental design are the use of structurally well defined DNA samples, making it possible to extend our knowledge of free radical reactions to understand the mechanisms of end product formation at low dose. By achieving the goals set down in this proposal, we will improve our ability to make risk/benefit decisions at low dose rates and low doses of radiation. Further, it provides the information needed to construct biologically relevant models of clustered DNA damage that can be used in studies of DNA repair and misrepair. The long-term objective of this proposed research program is to, (i) measure the types, yields and spatial distribution of the biologically relevant DNA lesions produced by the direct effect of ionizing radiation, (ii) determine the chemical mechanism(s) by which these DNA lesions are formed, and (iii) evaluate how molecules such as histones and other DNA binding proteins modulate the yield and distribution of these DNA lesions. Achievement of these objectives should make it possible to predict the composition and spatial distribution of the DNA lesions within biologically important clusters. This predictive capability is central to making risk/benefit decisions at both low dose rates and low doses of radiation and to constructing biologically relevant models of clustered DNA damage to be used by DNA enzymologist, biochemists and biophysicists in their DNA repair/misrepair studies.
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