A COMPONENT OF DNA DOUBLE-STRAND BREAK REPAIR IS DEPENDENT ON THE SPATIAL ORIENTATION OF THE LESIONS WITHIN THE HIGHER-ORDER STRUCTURES OF CHROMATIN

By the use of a modified neutral filter elution procedure variations i n the repair of DNA dsb have been observed between the ionizing radiat ion sensitive mutant xrs-5 and the parent cell line CHO-KI. Convention al neutral filter elution requires harsh lysis conditions to remove hi gher-order chromatin structures which interfere with elution of DNA co ntaining dsb. By lysing cells with non-ionic detergent in the presence of 2mol dm(-3) salt, histone-depleted structures that retain the high er-order nuclear matrix organization, including chromatin loops, can b e produced. Elution from these structures will only occur if two or mo re dsb lie within a single-looped domain delineated by points of attac hment to the nuclear matrix. Repair experiments indicate that in CHO c ells repair of dsb in loops containing multiple dsb are repaired with slow kinetics whilst dsb occurring in loops containing single dsb are repaired with fast kinetics. Xrs-5 cells are defective in the repair o f multiply damaged loops. This work indicates that the spatial orienta tion of dsb in the higher-order structures of chromatin are a possible factor in the repair of these lesions.