Knowledge of specific DNA base sequence damage and repair after ionizing radiation can provide clues to the molecular mechanisms by which radiation damage leads to mutation, carcinogenesis, and cell cycle disturbances relevant to radiation therapy. We can now get this kind of information because of advances in DNA base sequencing methods. The methods are readily applied to study of homogeneous DNA populations such as bacteriophage DNA and highly repetitive component Alpha of primate DNA (172 b.p. repeat). Alphoid DNA's are found in all human cells; they account for 25% of African green monkey cell DNA and their base sequences are known. Monkey cells from continuous lines BSC-1 and CV-1 will be irradiated and their Alpha sequences isolated using EcoR1*, Hind III, and Hae 111 endonuleases and agarose and polyacrylamide gel electrophoretic separation. After 5 feet 32p phosphate end-labeling and secondary restriction, the sites of strand scissions will be studied by the Maxam and Gilbert base sequencing method. There is no information yet available on DNA base sequence damage by ionizing radiation in living cells. Neverthesless, the techniques to learn this are available and the experiments are feasible. In vitro radiation studies on purified DNA were recently published; some preliminary base sequence damage has already been characterized. We proposed to learn the specific base sequence changes which are relevant in living cells. X-ray does-responses and the time course of in vivo repair of the base sequence damage will be studied, comparing this with damage induced under anoxic conditions. Synchronized HeLa and monkey cells will be irradiated and their progress through the cycle monitored by flow cytometry. Premutational base sequence changes which are not corrected after radiation can now be identified for the first time. Studies will be made on caffeine promoted escape from 2 arrest in irradiated cells, and on the progress of base sequence damage repair in the presence and absence of caffeine. Comparative studies with interspersed repetitive primate sequences, the Kpnl 1.2kb family, will be made because these newly discovered repetitive sequences are widely distributed; they are transcribed, and their base sequences and location in relation to specific human oncogenes are under study.
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