The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex

J Mol Biol. 2000 Dec 15;304(5):779-91. doi: 10.1006/jmbi.2000.4266.


Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Infective Agents / metabolism*
  • Bacteriophage T7 / enzymology*
  • Bacteriophage T7 / genetics
  • Base Sequence
  • Ciprofloxacin / metabolism
  • DNA / biosynthesis
  • DNA / genetics
  • DNA / metabolism*
  • DNA Replication*
  • DNA Topoisomerases, Type II / genetics
  • DNA Topoisomerases, Type II / metabolism*
  • DNA, Superhelical / genetics
  • DNA, Superhelical / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli
  • Multienzyme Complexes
  • Mutation
  • Nucleic Acid Synthesis Inhibitors*
  • Protein Binding
  • Templates, Genetic
  • Thioredoxins / metabolism


  • Anti-Infective Agents
  • DNA, Superhelical
  • DNA-Binding Proteins
  • Multienzyme Complexes
  • Nucleic Acid Synthesis Inhibitors
  • Thioredoxins
  • Ciprofloxacin
  • DNA
  • DNA-Directed DNA Polymerase
  • DNA Topoisomerases, Type II