The overall objective of this proposal is to determine the combined effect(s) of hyperthermia (40 to 48 degree) and radiation (0 to 100 Gy) exposure on DNA replication in mammalian cells, and the role of lesions in DNA replication in heat-, or combined heat and radiation-induced S phase cell cytotoxicity. The capacity of heated and/or irradiated S phase cells: to redistribute DNA being polymerized at the nuclear matrix into bulk DNA, to process this nascent DNA into higher- order (solenoid) chromatin structure, and ultimately to ligate this DNA fully into chromosome-sized DNA will be determined. The model we will test in heated cells is that long-term damage to the nuclear matrix, or its associated structures or macromolecules, results in residual lesions in DNA being replicated during hyperthermic exposure. A similar model will be tested to determine whether S phase cell sensitivity in the radiation sensitive ataxia telangiectasia human cells, the xrs CHO double strand break repair mutants or """"""""wild-type"""""""" cells exposed caffeine during X irradiation results from the failure of these cells to ligate replicating DNA fully into chromosomes. The nuclear alterations being examined in heated cells are the thermal denaturation of nuclear matrix protein(s), the precipitation of nuclear protein and RNA onto the nuclear matrix and the potentially irreversible association of the topoisomerase enzymes onto DNA adjacent to the nuclear matrix. We will determine whether these lesions in the DNA replicative process in heated cells result in the long-term presence of DNA strand breaks in DNA replicated during cell heating and thus ultimately to chromosome aberration formation and cytotoxicity.
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