The overall goal of this project is to provide an understanding of mechanisms of chromosome repair in mammalian cells and the relationship these processes may have to mutagenic and malignant processes. The approach taken is to isolate mutants from mammalian cell lines which are abnormally sensitive to killing by various mutagenic and carcinogenic agents and to investigate the genetic and biochemical basis for their sensitivity. A series of gamma ray-sensitive Chinese hamster ovary cell (CHO) mutants will be isolated using nylon cloth replica plating and dark-field photography. In this method, colonies are photographed 1 and 6 days after gamma radiation and the growth of colonies monitored by superimposing the first-day colony negative over the 6-day print. Colonies that continue to grow have a white halo surrounding a black center dot, whereas colonies with a reduced growth rate after gamma radiation have a much smaller or absent halo. Colonies are then isolated from the nylon cloth replicas and retested for sensitivity. Using a modified version of the above method, UV-sensitive CHO mutants will also be isolated. The suitability of methods for the direct isolation of DNA repair-defective mutants will also be investigated. Also, mutants which may be sensitive to killing by gamma- or UV-irradiation in specific phases of the cell cycle will be obtained from temperature-sensitive cell cycle mutants. As new mutants are isolated, they will be grouped into separate complementation groups and analyzed biochemically for defects in known DNA repair processes. Gamma ray damage induced at specific cell cycle phases may be repaired by different processes. This possibility will be investigated by correlating double strand DNA break repair with survival in a cell cycle-dependent gamma ray-sensitive mutant already isolated. A post-replication recovery mutant will be analyzed for the production of single strand DNA gaps opposite thymine dimers in newly synthesized DNA. The involvement of DNA repair in mutagenesis will be investigated by comparing the frequency of mutagen-induced mutation and the production of G6PD-colonies in repair-deficient mutants and parental cells. We feel certain these studies will provide important insights into the mechanisms of DNA repair and mutagenesis for gamma ray- and UV-light-induced damage in mammalian cells.
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