The long-term objective of this project is to provide an understanding of mechanisms involved in the repair of radiation induced DNA lesions in mammalian cells and how these processes may be involved in cellular survival to ionizing radiation. The goal of this specific proposal is to isolate a series of genetically distinct mutants from the Chinese hamster ovary cell line which are abnormally sensitive to killing by ionizing radiation and to characterize their sensitivity to this and other types of DNA damaging agents. Using nylon cloth replica plating and darkfield- photography, we will continue to isolate a series of gamma-ray- sensitive mutants. In a previously isolated mutant (XR-1) which is gamma-ray sensitive and repair deficient in the G1 phase of the cell-cycle but has normal resistance and repair capacity in late-S, we will isolate a double mutant which is sensitive in both G1 and S. Other mutants which are sensitive to killing by gamma-ray at specific phases of the cell-cycle will be isolated using temperature sensitive cell-cycle mutants, or by giving periodic doses of gamma-rays. Using the XR-1 cell as a positive control, we will investigate the possibility of developing methods for the direct isolation of mutants defective in the repair of gamma-ray- induced DNA strand breaks. As new mutants are isolated, they will be grouped into separate complementation groups by fusing different pairs of radiation sensitive mutants and testing the resulting hybrids for radiation resistance. Mutants belonging to different complementation groups will be examined for gamma ray sensitivity throughout the cell-cycle to determine whether they are unusually sensitive in a particular phase of the cell- cycle. The mutants' sensitivity to other types of DNA damaging agents will also be examined. We believe that a collection of genetically distinct radiation-sensitive mutants will prove to be invaluable tools in understanding the cellular processes responsible for survival to ionizing radiation.
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