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DNA double-strand breaks induced by reactive oxygen species promote DNA polymerase IV activity in Escherichia coli

Sarah S. Henrikus,Camille Henry,J. McDonald,Yvonne Hellmich,Steven T. Bruckbauer,Megan E Cherry,E. Wood,R. Woodgate,M. Cox,Antoine M. van Oijen,Harshad Ghodke,Andrew Robinson

Published 2019 in bioRxiv

ABSTRACT

Under many conditions the killing of bacterial cells by antibiotics is potentiated by damage induced by reactive oxygen species (ROS). In most bacteria, ROS primarily target biomolecules such as proteins and DNA. Damage to DNA, particularly in the form of double-strand breaks (DSBs), is a major contributor to cell death. DNA polymerase IV (pol IV), an error-prone DNA polymerase produced at elevated levels in cells experiencing DNA damage, has been implicated both in ROS-dependent killing and in DSB repair (DSBR). Here, we show using single-molecule fluorescence microscopy that ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the DNA-damaging antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in intracellular pol IV concentration that is strongly dependent on both ROS and DSBR. Second, in cells that constitutively express pol IV, co-treatment with a ROS mitigator dramatically reduces the number of DSBs as well as pol IV foci formed, indicating a role of pol IV in the repair of ROS-induced DSBs. Significance Many antibiotics induce an accumulation of reactive oxygen species (ROS) in bacterial cells. ROS-induced damage to DNA, in particular formation of double-strand breaks (DSBs), potentiates killing by several bactericidal antibiotics. Here we used single-molecule fluorescence microscopy to reveal new links between ROS-induced DSBs and the activity of error-prone DNA polymerase IV (pol IV). We found that antibiotic-induced up-regulation of pol IV production requires active formation of DSB intermediates and can be supressed by ROS mitigators. The formation of pol IV foci, which reflect DNA-binding events, also requires DSB repair. Our findings support a major role for pol IV in DSB intermediates and reveal new details of how antibiotic treatment can potentially drive the development of antibiotic resistance in bacteria.

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