RADIATION DAMAGE IN DNA

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This is the second revised application of CA46838 "Analysis of Radiation Damage in DNA". Twenty-five different lesions have been identified in intact DNA oligomers. High resolution HPLC has served to isolate damaged oligomers bearing different lesions. The identification of these lesions has been accomplished through the use of NMR spectroscopy, complemented by fast atom bombardment (FAB) mass spectrometry. Additional lesions would be identified, especially in irradiated tetramers and hexamers. Preliminary studies have revealed stark differences between the HPLC product profile from irradiated single-stranded oligomer and the product profile from the same oligomer irradiated in duplex form. The double-stranded form manifestly interferes with the formation of particular lesions. An objective of the continuing research would be to identify these lesions. Radiation-modified DNA oligomers constitute an important resource. The behavior of a DNA oligomer bearing a specific lesion as substrate to endonucleases, kinases and polymerases can be studied. For example, it has been demonstrated in this laboratory that many radiation-induced lesions inhibit nuclease Pl. This phenomenon led to the design of a sensitive assay for the detection of Pl-resistant lesions in DNA. The assay has been implemented for the so-called formamido lesion. The assay would be further refined and extended to the analysis of DNA taken from exposed cells. The assay would also be extended to other lesions many of which are associated with oxidative stress as well as radiation damage. Another objective is to associate with each specific lesion a measure of the propensity of that lesion to induce mutation. An in vitro assay is proposed to evaluate the effect of the lesion on the -function of DNA polymerase. Again, the basic resource for the proposed experiments are oligomers bearing a specific lesion which are to be used as substrate for testing polymerase function.