MUTATIONS OF VACCINIA VIRUS-DNA TOPOISOMERASE-I THAT STABILIZE THE CLEAVAGE COMPLEX

Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli ex pressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to d etermine the mechanism of SOS induction. The K167D mutant enzyme had r educed binding affinity for the DNA substrate with a K(app) that was 1 0-fold higher than wild type. Nevertheless, in reactions with high enz yme concentration, its substrate cleavage activity was 90% that of wil d type. The G226N mutant enzyme had virtually wild type binding and cl eavage activities. However, intermolecular religation by these two mut ants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants i n vivo induce the SOS response in E. coli due to the shift of topoisom erase cleavage-religation equilibrium towards cleavage and increased s tability of the cleavage complex. The mutation thus has a similar effe ct as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents.