Kinetics of DNA Double-strand Break Repair Throughout the Cell Cycle as Assayed by Pulsed Field Gel Electrophoresis in CHO Cells

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Abstract
Repair of DNA double-strand breaks (dsb) was measured in exponentially growing, plateau-phase and synchronized G1, G1/S, early S, mid-S, late S, G2 + M and mitotic CHO cells. Cells were exposed to 50 Gy X-rays and allowed to repair (up to 4h) in suspension at 37°C. The dsb rejoining was measured by means of asymmetric field inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis technique. The fraction of DNA-associated [14C]thymidine activity released from the plug (FAR) during electrophoresis was used as a parameter to determine the number of dsb present in the DNA. The assay had been previously calibrated using 125I-decay and its sensitivity in detecting dsb throughout the cell cycle established. Biphasic kinetics of dsb rejoining with a fast and a slow component were obtained throughout the cell cycle, including mitosis, as well as in the various stages of growth. Repair kinetics were described mathematically as the sum of two exponential functions. The repair half-times calculated for the fast component by least-square fitting to the data were in the range of 7–14 min, with the shorter values reached in mid-S and late S and the longer ones during G1 and mitosis. The repair half-times of the slow component were in the range between 60 and 90 min and did not show consistent fluctuations throughout the cell cycle. The fraction of dsb repaired by the slow component ranged from 18% to 37% and did not show consistent variation throughout the cell cycle either. These results suggest that the state of chromatin condensation has only a limited impact on the ability of the cells to rejoin dsb, and indicate that the cell cycle-dependent fluctuations in radiosensitivity cannot be explained by alterations in the rate of rejoining of dsb. The repair half-times of the slow component of dsb rejoining were similar to the half-times of rejoining of chromosome breaks as visualized by the technique of premature chromosome condensation, suggesting a cause-effect relationship between rejoining of this subject of dsb and rejoining of chromosome breaks.

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