The primary goal of this project is to elucidate the effect of thiols on the free radical processes that occur in DNA after irradiation under anoxic and oxic conditions. These studies will test three models for the chemical oxygen enhancement effect and will investigate free radical mechanisms for thiol radioprotection. The studies will employ electron spin resonance spectroscopy and HPLC and GC-mass spectroscopy and are performed under conditions that emphasize the direct effect of radiation. Through the irradiation of frozen anoxic and oxygenated DNA-thiol solutions and the careful analysis of electron spin resonance spectra the free radical mechanisms involved in the transfer of DNA anionic, cationic and neutral radical sites to thiols of differing charge will be elucidated. HPLC and GC-MS will be employed to identify DNA and thiol products in the samples analyzed by ESR. The product analyses will test the mechanisms elucidated by ESR. From this analysis the role of O2 in reactions with DNA radicals and with thiol radicals will be determined. The primary DNA radicals which react most readily with thiols, and the identity of the thiol radical intermediates produced in these reaction will be determined. Reactions of sulfoxyl radicals with DNA will be searched for. The role of the disulfide anion radical in the chemical oxygen enhancement effect will be elucidated by an investigation of a series of disulfides. Individual DNA base ion radicals will be formed on nucleotides and their reaction with thiols investigated in order to elucidate the efficiency of radical transfer from each of the various bases. At a later stage of work on these model systems polynucleotides and oligonucleotides will also be investigated. The role of histones on the radical transfer process to charged thiols will be investigated as well. Polyamines such as spermine will also be employed as competitive binding agents. Changing the relative concentration of thiol/binding species will provide further insight into the importance of thiol binding to radical transfer and act as a model for the effect of histone-DNA interactions on radical transfer to thiols.
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