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

Abstract

Under many conditions the killing of bacterial cells by antibiotics is potentiated by DNA damage induced by reactive oxygen species (ROS)1–3. A primary cause of ROS-induced cell death is the accumulation of DNA double-strand breaks (DSBs)1,4–6. 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 DSBR7–15. Here, we show using single-molecule fluorescence microscopy that ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in intracellular pol IV concentrations that is strongly dependent on both ROS and DSBR. Second, in cells that constitutively express pol IV, treatment with an ROS scavenger dramatically reduces the number of pol IV foci formed upon exposure to antibiotics, indicating a role for pol IV in the repair of ROS-induced DSBs.

Modulation of DNA polymerase IV activity by UmuD and RecA* observed by single-molecule time-lapse microscopy

“…The dnaX-mKate2 allele encodes for a fluorescent fusion of the τ clamp loader protein, serving as a marker for the replisome, τ-mKate2. We previously showed that the fluorescent protein fusion of DinB-YPet is fully functional, yielding pol IV-dependent mutagenesis activity upon ciprofloxacin treatment in both dnaX + and dnaX-mKate2 cells (Henrikus et al ., 2018b; Henrikus et al ., 2019b).…”

“…Ciprofloxacin is a DNA gyrase inhibitor, which generates DSBs (Zhao et al ., 1997) and rapidly halts DNA synthesis (Deitz et al ., 1966; Snyder and Drlica, 1979). Defects in DSB processing strongly suppress both pol IV up-regulation and focus formation (Henrikus et al ., 2019b). Interestingly, in vitro , pol IV is capable of associating with RecA(E38K)-ATPγS filaments formed on dsDNA.…”

“…A series of live-cell studies indicate that pol IV operates in the repair of double-strand breaks (DSBs) (Ponder et al ., 2005; Foster, 2007; Shee et al ., 2011; Rosenberg et al ., 2012; Shee et al ., 2012b; Mallik et al ., 2015; Moore et al ., 2017). Reducing DSB formation (by mitigating the destructive effects of reactive oxygen species) or introducing defects in the end-resection of double-strand breaks (Δ recB mutation) greatly reduces the number of pol IV foci formed in cells treated with ciprofloxacin or trimethoprim (Henrikus et al ., 2019b). At end-resected DSBs, RecA nucleoprotein filaments facilitate repair through homologous recombination (Cox, 2007), suggesting that pol IV should colocalise with RecA* structures in cells engaged in DSB repair.…”

Potential link between selection for high GC content and repair of double strand breaks in prokaryotic genomes

“…Much debate has resulted over 14 the relative contribution of these two mechanisms to the observed genomic GC 15 content in prokaryotes [8,9,5,4,7,10]. 16 This debate between proponents of the selection and BGC hypotheses con-17 tinues, with many studies focusing on patterns of genetic diversity that by them- 18 selves cannot easily differentiate between these two hypotheses because recom- 19 bination will also locally increase the efficiency of selection [11,9]; however, 20 the addition of phenotypic information provides the tantalizing clue that GC 21 content correlates with shared environmental factors [2,3,12,13] independent 22 of phylogenetic similarity [3]. Thus, these environmental factors must either 23 lead to an unknown selective advantage for high/low GC content [11] or lead to 24 elevated rates of BGC through an as-yet unknown mechanism.…”

“…25 We noticed that many environments containing high GC content microbes, 26 such as soils and aerobic environments [3,12], induce relatively high rates of 27 DNA damage in the form of double-strand breaks (DSB) that necessitate repair 28 [14,15]. For instance, in aerobes, this damage typically results from reactive 29 oxygen species produced during metabolism [14] that can lead to DSBs by pro- 30 ducing collapsed replication forks [16], as well as via a number of other mecha-31 nisms (often in conjunction with other stressors; [17,18,19,20,21,22,23,24]). 32 In soil-dwelling microbes, DSBs are associated with desiccation and spore for- 33 mation [25,26,27,28].…”