Quinolones are widely prescribed, broad‐spectrum antibacterial drugs. Despite their widespread use, the molecular mechanisms of quinolone cytotoxicity are not clearly understood. Quinolones target topoisomerases, preventing the resealing of otherwise transient DNA breaks. Poisoned topoisomerase, or stabilized cleavage complexes, are not sufficient for cell death. The goal of this study is to determine how stabilized cleavage complexes are converted into irreversible double strand DNA breaks (DSBs) that accumulate and drive quinolone cytotoxicity.Previous studies identified an unexpected connection between dnaQ and quinolone‐induced DNA damage in Escherichia coli. The ɛ subunit of DNA polymerase III, encoded by dnaQ, is an exonuclease providing the 3′ ‐> 5′ proofreading activity of the core. The absence of ɛ causes higher mutation rates, slow growth, constitutive SOS, and a defective SOS response following quinolone treatment. We found that these phenotypes are separable, which implies that ɛ has multiple, separable roles, aside from proofreading.We explore the role of ɛ‐β interaction in quinolone‐induced DSB generation. Using two different types of cell lysis in conjunction with pulsed‐field gel electrophoresis, we distinguished between latent breaks at the cleavage complex and overt chromosomal breaks due to additional processing. We provide evidence that supports the replication run‐off model of DSB generation, whereby a stronger ɛ‐β interaction causes the replicase to physically interact with stabilized cleavage complexes rather than stalling or disassembling.Support or Funding InformationGettysburg College Research & Professional Development Grant & Howard Hughes Medical Institute ‐ Cross‐Disciplinary Sciences Institute at Gettysburg College