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 to cell death. We show here that an active strand breakage and reunion activity is required for formation of a norfloxacin-topoisomerase IV-DNA ternary complex that can arrest the progression of replication forks in vitro. Mutant topoisomerases containing either an active site mutation, 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 replication 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 likely results from a two-step process: (i) conversion of the frozen topoisomerase-quinolone-DNA ternary complex to an unreversible form; and (ii) generation of a double-strand break by subsequent denaturation of the topoisomerase, perhaps by an aborted repair attempt.
Hiasa
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Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Yousef
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Marians
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KJ
D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D016297:Mutagenesis, Site-Directed; D009643:Norfloxacin / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D015363:Quinolones / Q000378:metabolism*
D015363:Quinolones; D004247:DNA; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II; D009643:Norfloxacin