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 in vivo. Direct effects accounts for 30-50% of the in vivo DNA damage for low LET (linear energy transfer) radiations. At high LET, direct effects account for about 80% of the damage. Knowledge of the mechanism by which ionizing radiation produces unrepaired damage in DNA is critical in determining the risks, such as induction of cancer of leukemia, due to low dose and low dose rates of radiation. Achievement of the stated objective will be a major benefit in risk assessment and disease treatment.
The specific aims examine free radical processes initiated in DNA by direct ionization.
These aims are: 1) to determine the effect of base sequence and packing on free radical trapping in DNA, 2) to form a mechanistic link between diamagnetic damage and free radical intermediates, and 3) to determine the boundaries in the hydration layer for electron and hole transfer to DNA. The approach is to use electron paramagnetic resonance spectroscopy (EPR) to study free radical intermediates formed in oligodeoxynucleotides and DNA. Oligodeoxynucleotides will be studied in single crystal and polycrystalline form and DNA will be studied in the form of films. EPR measurements will be made at 4K to maximize detection sensitivity. Samples will be irradiated at 4K, 77K, 200K, 270K, and 300K using 70 kV X-rays. In the analysis of stable end products, strand breaks will be identified using high performance liquid chromatography, release of free base and base damage will be determined using gas chromatography/mass spectrometry, and the structure of modified oligodoxynucleotides will be determined using NMR> A major difficulty in determining the effects of hydration and molecular packing on damage formation in DNA polymers is the lack of precise knowledge of packing in condensed systems. These difficulties will be overcome by using crystalline oligodoxynucleotides for which the structures have been determined by X-ray crystallography. He will investigate crystals of d(CGATCGATCG), d(CCAACGTTGG), d(CTCTCGAGAG), d(CTCTGAG), and the d(CGATCG):daunomycin complex. These experiments will be instrumental in determining which free radical intermediates lead to specific types of stable diamaonetic products in DNA damaged directly by low LET radiation and learning how the progression of damage is effected by base sequence and packing.
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