Interactions between DNA helicases and frozen topoisomerase IV-quinolone-DNA ternary complexes

Collisions between replication forks and topoisomerase-drug-DNA ternary com plexes result in the inhibition of DNA replication and the conversion of th e normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell deat h, To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes, A strand displaceme nt assay was employed to assess the effect of topoisomerase IV (Topo IV)-no rfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-anti gen, and UvrD DNA helicases, The ternary complexes inhibited the strand dis placement activities of these DNA helicases. Unlike replication fork arrest , however, this general inhibition of DNA helicases by Topo IV-norfloxacin- DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases, UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen d id not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not suf ficient to generate cytotoxic DNA lesion.