Ionizing radiation is widely utilized in cancer therapy. While some cancers have been treated successfully, an unacceptable fraction of treatments still fail. The ensuing recurrences and secondary tumors are often more aggressive than the original, possibly because of treatment-induced mutations. These mutations are not well defined due to the varied types of DNA damage ionizing radiation, the randomness of the damage, and its relative infrequency in the genome. Recent advances in nucleic acid chemistry make it possible to synthesize many of the types of damage detected in DNA subjected to ionizing radiation. These modified nucleotides can be incorporated into specific sites in cloned genes by standard molecular biology techniques. The proposed work will utilize this combined approach to place types of DNA damage, characteristic of ionizing radiation, at designated sites in a cloned DNA fragment. We will then determine how such damage affects eukaryotic gene expression and gene integrity. DNA damage will be positioned in synthetic oligonucleotides corresponding to transcription factor binding sites. A gel shift assay will be utilized to determine the binding and stability of transcription complexes containing either normal or damaged DNA elements. Damage will also be incorporated into specific sites in a synthetic DNA template and transcribed in vitro with a nuclear extract. The effect of specific types of DNA damage of gene transcription and fidelity will be determined. Related experiments will also determine if transcription transforms two opposed single-strand breaks into a more lethal double-strand break. Finally, we will measure the relative stability of duplex oligonucleotides containing base damage sites using the competitive mobility shift assay and determine whether a stability correlates with the polymerase by-pass or mutagenicity results obtained from the in vitro transcription studies.
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