The overall long-term goal of this research program is to investigate the repair of the non-dimer DNA damages induced by the UV component of sunlight. The study of these non-dimer DNA damages is of importance as they appear to be of biological significance in cells exposed to solar UV radiation. The UV wavelengths present in sunlight at the earth's surface represent an important health concern as this region of sunlight appears to be responsible for the induction of most skin cancers, which is the most common form of cancer in the United States. It is therefore essential for the understanding of skin carcinogenesis, that the mechanisms by which cells respond to DNA damages induced by solar UV radiation be studied. In the previous project period mutant cell lines were obtained, using the ICR 2A cell line, that are hypersensitive to the solar UV-induction of non-dimer DNA damages. Therefore, the specific aims of the first section of this project involve an examination of various aspects of repair in the solar UV-sensitive cell lines compared with the parental ICR 2A cells. First, the overall level of excision repair will be measured in solar UV-irradiated cells using the modified bromodeoxyuridine photolysis assay that was developed as part of this project. Next, the inhibition and recovery of semiconservative DNA synthesis will be examined in these cells following exposure to solar UV using the step alkaline elution assay. The following specific aim involves the measurement of the repair of the major non-dimer damages induced by solar UV irradiation. The first three lesions will be DNA strand breaks, DNA-protein crosslinks and endonuclease III-sensitive sites. These damages will all be measured using the alkaline elution assay. In addition, DNA-protein crosslinks will be quantitated using the nitrocellulose filter binding assay. The next specific aim involves an extension of work performed int eh previous project period in which it was found that DNA-protein crosslinks form upon incubation of cells following solar UV-irradiation. This work will involve the identification of these protein(s) using both the SDS-potassium technique and nitrocellulose filter binding. The protein(s) that are isolated will then be analyzed using SDS-polyacrylamide gel electrophoresis and Western blotting. Finally, experiments will be performed with the goal of cloning the human genes that are capable of correcting the deficiencies in these solar UV sensitive mutants.
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