An antitumor drug-induced topoisomerase cleavage complex blocks a bacteriophage T4 replication fork in vivo

Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trappi ng covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexe s themselves are not sufficient to cause cell death. Rather. active cellula r processes such as transcription and/or replication are probably necessary to transform cleavage complexes inter cytotoxic lesions. Using defined pla smid substrates and two-dimensional agarose gel analysis, we examined the c ollision of an active replication fork with an antitumor drug-trapped cleav age complex, Discrete DNA molecules accumulated on the simple Y are, with b ranch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid, Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-f orm DNA appeared even in the absence of two important phage recombination p roteins, implying that Y-form DNA is the result of replication rather than recombination, This is the first direct evidence that a drug-induced topois omerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisome rase cleavage site, implying that the replication complex was inactivated ( at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA les ions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DIVA damage.