Direct Measurement by Pulsed-field Gel Electrophoresis of Induction and Rejoining of X-ray-induced Double-strand Breaks in Cultured Mouse Cells

Abstract

The induction and rejoining of X-ray-induced double-strand breaks (dsb) in chromosomal DNA has been difficult to measure. We have developed a pulsed-field gel electrophoresis (PFGE)-based system for directly estimating DNA sizes between 0·2 and 10 million base pairs. With this system we can estimate average DNA sizes from randomly broken chromosomes by measuring the approximate molecular weight of the maximum DNA concentration. In practice this is effective where the average is between 1 and 4 million bp allowing both shoulders of the distribution to be observed. This corresponds to a dose range of 20–80 Gy. Qualitative differences from non-irradiated DNA can be observed down to about 5 Gy. We have confirmed the dose response by utilizing methotrexate-resistant mouse cells containing circular double-minute (dm) chromosomes of 1, 1·5, and 3 million bp. The kinetics of dsb rejoining from doses of 50 and 5 Gy was investigated: 50 Gy reduced the chromosomal DNA to an average size of approximately 1 million bp, followed by a constant repair rate of 44 dsb per minute per cell for 3 h (assuming a total genome size of 10 million bp).