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Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded

Jennifer Reineke Pohlhaus1, Kenneth N Kreuzer

  • 1Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

Molecular Microbiology
|May 27, 2005
PubMed

Insights

Antibacterial quinolones stall bacterial DNA replication forks by forming stable gyrase-DNA complexes. These stalled forks can lead to DNA double-strand breaks through indirect mechanisms, impacting bacterial survival.

Area of Science:

  • Molecular Biology
  • Bacteriology
  • Genetics

Background:

  • Antibacterial quinolones target type II DNA topoisomerases, stabilizing enzyme-DNA complexes.
  • These complexes are implicated in generating double-strand DNA breaks during replication.

Purpose of the Study:

  • To investigate the interaction between replication forks and quinolone-induced gyrase-DNA cleavage complexes in Escherichia coli.
  • To provide in vivo evidence for replication fork stalling by these complexes.

Main Methods:

  • Utilized plasmid pBR322 and two-dimensional agarose gel electrophoresis.
  • Examined stalled replication forks under varying DNA extraction conditions (resealing vs. cleavage).

Main Results:

  • Observed stalled replication forks, evidenced by bubble arcs and discrete spots on gels.
  • Demonstrated that quinolone-induced gyrase-DNA complexes directly block replication forks.
  • Identified stalled forks both with and without bound cleavage complexes, suggesting gyrase dissociation.

Conclusions:

  • Quinolone-induced cleavage complexes are potent inhibitors of bacterial replication forks in vivo.
  • Replication fork stalling can occur even after gyrase dissociation.
  • A model for indirect double-strand break formation following quinolone treatment is proposed.

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