INDUCTION OF DNA DOUBLE-STRAND BREAKS BY RESTRICTION ENZYMES IN X-RAY-SENSITIVE MUTANT CHINESE-HAMSTER OVARY CELLS MEASURED BY PULSED-FIELDGEL-ELECTROPHORESIS

This investigation was designed to determine whether the cytotoxic eff ects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hams ter ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and Ba mHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-held gel electrophoresis (PFGE). Two form s of PFGE were employed: asymmetric field-inversion gel electrophoresi s (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repa ir deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant invo lves the rejoining of DSBs. Although the cutting frequency was directl y related to the length of the recognition sequence for four restricti on enzymes, there was no simple correlation between the cytotoxic effe ct and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting se quence itself map play a role in restriction enzyme-induced cell killi ng. (C) 1995 by Radiation Research Society