Examining x-ray damage and repair of cellular DNA at the base sequence level required obtaining homogeneous populations of DNA sequences from the irradiated cells. We demonstrated the feasibility of this in studies on highly repetitive component alpha DNA sequence prepared from irradiated monkey CV-l cells. The system is being used to evaluate the time course of repair of alpha DNA and the influence of caffeine on repair. We now extend the studies to alpha DNA residing in irradiated plasmids transfected into host cells. This approach permits studies on x-ray damage and repair of homogeneous populations cloned genes. Eventually, it should provide a host cell react- ivation system for detecting and evaluating the activity of individual DNA repair enzymes and the transfected genes which code for them. Plasmids serve as surrogate targets for cellular DNA. Damage to plasmid sequences are verified before transfection; the time course of repair is followed, in unirradiated cells, thereby avoiding interference from radiation damage to cell functions. With the host cell reactivation scheme, residual DNA strand and base damage will be scored at the base sequence level in cells of repair proficient and repair deficient constitution (e.g., AT and XP). The topology of plasmids carrying x-ray damaged DNA sequences must be taken into account, as in our report on expression of genes coding for chloramphenicol acetyltransferase in cells transfected with pSV2CAT. Information on the relationship of plasmid form and the repair of specific sequences can now be attained. Together with data on x-ray inactivation of gene expression, the studies we propose with circular plasmids provide a model for studying x-ray effects on DNA in the chromatin, where the DNA is also arranged in closed loops. Residual unrepaired damage in sequences transfected into AT cells are of special interest because certain DNA repair deficiencies in AT are believed to be present in as many as 5% of all persons dying of cancer before age 45 years.
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