ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL MUTANTS

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DESCRIPTION: Based on previous work showing that a "ku-like" complex of proteins in mammalian cells binds to double strand breaks, but is absent in a repair-deficient Chinese hamster mutant, xrs, the long term goal is to test the hypothesis that this "ku-like" complex plays some role in regulating gene transcription after DNA damage, protect DNA ends from nuclease digestion, affect DNA repair and have helicase, exonuclease kinase and topoisomerase activities, as well as to have the ability to insert single strand DNA into duplex DNA. This hypothesis, which is formulated to explain the biochemical basis of cellular resistance to cell killing by ionizing radiation, will be tested via several specific aims: (a) to identify proteins associated with a putative DNA repair complex; and (b) to investigate the biological function of the complex. To identify the proteins associated with the complex, proteins will be purified, analyzed by a combination of 2D diagonal gels and Western blotting using antibodies to known proteins known or suspected to be associated with the hypothesized function of the complex. Partial sequence analysis will be done on proteins not identified with known antibodies, cDNA clones isolated by screening libraries with sequences generated from the unknown peptides will be sequenced and compared to known sequences. Ku antibodies will also be used to immunoprecipitate associated proteins in both CHO and human cells. In addition a modified mobility shift assays, a series of repair deficient hamster mutants will be examined for abnormalities in the "ku-like "complex. Electron microscopy will be used to determine if end-to- end association of DNA by this complex occurs.