The polymer poly(ADP-ribose) is present is the nuclei of all eukaryotic organisms. While a number of studies have suggested the involvement of this polymer in the repair of damage produced by ionizing radiation and other agents, the biological role of this polymer in DNA repair still remains unclear. Thus, the ultimate goal of this research is to determine what the nature of this involvement might be in mammalian cells. To answer this question, we propose to isolate mutants in the Chinese hamster ovary cell which are deficient in the synthesis of poly(ADP-ribose) and determine the physiologic and biochemical consequences of this deficiency. These mutants will be isolated using a non-selective screening approach in which colonies are examined in situ for enzyme activity using radioactive substrate (NAD) and autoradiography. Viable cells from putative enzyme-deficient colonies will be recovered from nylon cloth replicas and tested for poly(ADP-ribose) enzyme activity in cell free extracts. Poly(ADP-ribose) polymerase negative mutants will be examined for abnormal sensitivity to killing by gamma-rays and other DNA damaging agents. Those agents which produce an increase in sensitivity will also be examined for sensitivity through the cell- cycle. The kinetics of repair of single-and double-strand DNA breaks will be examined for those agents which produced increased sensitivities. Also repair replication and unschedule DNA synthesis will be examined. We believe that biochemical characterization of a poly(ADP)-ribose-deficient mutant offers the prospect of elucidating the role of this polymer in DNA repair.
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