DNA STRAND CLEAVAGE IS REQUIRED FOR REPLICATION FORK ARREST BY A FROZEN TOPOISOMERASE-QUINOLONE-DNA TERNARY COMPLEX

The formation of a topoisomerase-quinolone-DNA ternary complex leads t o cell death, We show here that an active strand breakage and reunion activity is required for formation of a norfloxacin-topoisomerase IV-D NA ternary complex that can arrest the progression of replication fork s in vitro, Mutant topoisomerases containing either an active site mut ation, a quinolone resistance-conferring mutation, or both, could all bind DNA as well as the wild type, but unlike the wild-type, could not halt replication fork progression, The collision between the replicat ion fork and the frozen topoisomerase converted the cleavable complex to a nonreversible form but did not generate a double-stranded break. Thus, the cytotoxicity of this class of topoisomerase inhibitors likel y results from a two-step process: (i) conversion of the frozen topois omerase-quinolone-DNA ternary complex to an unreversible form; and (ii ) generation of a double-strand break by subsequent denaturation of th e topoisomerase, perhaps by an aborted repair attempt.