The long-term objective of this project is to determine the mechanisms by which ionizing radiation, through direct effects, alters the primary structure of DNA. Damage due to direct effects accounts for 20-30% of the total damage to DNA for low LET radiations. At high LET, it increases to 80% of the damage. The accumulation of unrepaired damage in DNA is a critical variable in determining the risks due to long-term exposures to low doses of radiation, risks such as induction of cancer or leukemia. Achievement of the stated objective will be a major benefit in risk assessment and disease treatment.
The specific aims work toward the long-term goal by examining free radical processes initiated in DNA by direct ionization.
These aims are: (1) to determine the relative electron trapping efficiencies of the DNA bases and the spatial distribution of the electron adducts, (2) likewise, to determine the sites and distribution of electron loss, (3) to determine the yields of these initial free radical products as a function of chemical environment and molecular structure, working toward a chromatin environment, and (4) to follow the free radical reactions, qualitatively and quantitatively, that connect the initial free radical products with more stable end products. The approach is to use electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) spectroscopies to study DNA constituents, deoxyoligonucleotides, deoxypolynucleotides, and DNA. These materials are studied in the form of powders, crystals, and frozen glasses. By X-irradiating at low temperatures, 4K, the initial free radical products are stabilized; controlled warming makes it possible to observe the sequence of free radical reactions that lead to more stable end products. The main thrust will involve experiments that characterize each of the one-electron reduced bases and then use these characteristics to determine the sites of electron attachment in complementary deoxyoligonucleotides of known base sequence and known conformation. Experiments that follow will apply this approach to oxidized instead of reduced bases. The deoxyoligonucleotides will be studied as solutes dissolved in 12 M LiCl glasses. Single crystal ESR/ENDOR will be used to assist in free radical characterization and in identifying the critical variables governing free radical yield. Powder ESR will be used in extending the information obtained from crystals and glasses to DNA polymers.
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