Repair of topoisomerase-mediated DNA damage in bacteriophage T4

Type II topoisomerase inhibitors are used to treat both tumors and bacteria l infections. These inhibitors stabilize covalent DNA-topoisomerase cleavag e complexes that ultimately cause lethal DNA damage. A functional recombina tional repair apparatus decreases sensitivity to these drugs, suggesting th at topoisomerase-mediated DNA damage is amenable to such repair. Using a ba cteriophage T4 model system, we have developed a novel in vivo plasmid-base d assay that allows physical analysis of the repair products from one parti cular topoisomerase cleavage site. We show that the antitumor agent 4 '-(9- acridinylamino)-methanesulphon-m-anisidide (m-AMSA) stabilizes the T4 type II topoisomerase at the strong topoisomerase cleavage site on the plasmid, thereby stimulating recombinational repair. The resulting m-AMSA-dependent repair products do not form in the absence of functional topoisomerase and appear at lower drug concentrations with a drug-hypersensitive topoisomeras e mutant. The appearance of repair products requires that the plasmid conta in a T4 origin of replication. Finally, genetic analyses demonstrate that r epair product formation is absolutely dependent on genes 32 and 46 largely dependent on genes uvsX and uvsY, and only partly dependent on gene 49. Ver y similar genetic requirements are observed for repair of endonuclease-gene rated double-strand breaks, suggesting mechanistic similarity between the t wo repair pathways.