THE INDUCTION AND REVERSAL OF TOPOISOMERASE-II CLEAVABLE COMPLEXES FORMED BY NUCLEAR EXTRACT FROM THE CHO DNA-REPAIR MUTANT, XRS1

The gamma-ray sensitive CHO cell mutant xrs1 is hypersensitive to antitumour drugs that stabilise DNA topoisomerase II (topoII) cleavable complexes. Sensitivity appears to result from DNA double-strand breaks (DSBs) that persist in xrs1 cells, but not wild-type CHO-K1 cells, following drug removal. One possible explanation for the persistence of DSBs in xrs1 cells is a defect in topoII which reduces its ability to reseal the DSBs associated with cleavable complexes following drug removal. To address this possibility, cleavable complexes formed in vitro by incubating VP16, plasmid DNA and nuclear extract from either CHO-K1 or xrs1 cells were induced to reverse by adding EDTA or salt to the reaction, or by raising the temperature to 65-degrees-C, or by dilution of the drug. The fraction of drug-induced cleavable complexes that reversed in these experiments was dependent on how reversal was induced, and ranged from 55 to 95%. However, the extent of reversal was independent of the source of nuclear extract in all of the experiments, indicating that CHO-KI and xrs1 topoII is equally able to reseal complex-associated DSBs during cleavable complex reversal in vitro.