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DNA Double-Strand Breaks Induced by Reactive Oxygen Species Promote bioRxiv preprint doi: https://doi.org/10.1101/533422; this version posted January 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 DNA double-strand breaks induced by reactive oxygen 2 species promote DNA polymerase IV activity in 3 Escherichia coli 4 5 Sarah S. Henrikus1,2, Camille Henry3, John P. McDonald4, Yvonne Hellmich5, 6 Elizabeth A. Wood3, Roger Woodgate4, Michael M. Cox3, Antoine M. van Oijen1,2, 7 Harshad Ghodke1,2, Andrew Robinson1,2* 8 1Molecular Horizons Institute and School of Chemistry and Biomolecular Science, University 9 of Wollongong, Wollongong, Australia 10 2Illawarra Health and Medical Research Institute, Wollongong, Australia 11 3Department of Biochemistry, University of Wisconsin-Madison, United States of America 12 4Laboratory of Genomic Integrity, National Institute of Child Health and Human 13 Development, National Institutes of Health, Bethesda, Maryland, United States of America 14 5Institute of Biochemistry, Goethe Universität, Frankfurt, Germany 15 *Corresponding author. Mailing address: School of Chemistry, University of Wollongong, 16 Wollongong, NSW 2500, Australia. Email: [email protected]. 17 1 bioRxiv preprint doi: https://doi.org/10.1101/533422; this version posted January 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Under many conditions the killing of bacterial cells by antibiotics is potentiated by 2 DNA damage induced by reactive oxygen species (ROS)1–3. A primary cause of ROS- 3 induced cell death is the accumulation of DNA double-strand breaks (DSBs)1,4–6. DNA 4 polymerase IV (pol IV), an error-prone DNA polymerase produced at elevated levels in 5 cells experiencing DNA damage, has been implicated both in ROS-dependent killing 6 and in DSBR7–15. Here, we show using single-molecule fluorescence microscopy that 7 ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the 8 antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in 9 intracellular pol IV concentrations that is strongly dependent on both ROS and DSBR. 10 Second, in cells that constitutively express pol IV, treatment with an ROS scavenger 11 dramatically reduces the number of pol IV foci formed upon exposure to antibiotics, 12 indicating a role for pol IV in the repair of ROS-induced DSBs. 13 Two mechanisms for ROS-induced DSB formation have been proposed (Figure 1a). 14 The first invokes oxidisation of the cellular nucleotide pool, leading to increased 15 incorporation of oxidised nucleotide triphosphates (e.g. 8-oxo-dGTP) into the DNA by DNA 16 polymerases4,7,16. Subsequent initiation of base-excision repair (BER) creates single- 17 stranded DNA (ssDNA) gaps. In cases where BER is initiated at nearby sites, DSBs may be 18 formed1,4,7. Evidence for a second mechanism of ROS-dependent DSB formation has 19 emerged from a recent mechanistic study of thymineless death in Escherichia coli5. The 20 ROS-driven potentiation of killing by both antibiotic treatment and thymineless death can be 21 abrogated through the addition of ROS scavengers to the culture medium5,17,18. Using 22 microscopy to quantify ssDNA gaps and DSBs in cells undergoing thymineless death, Hong 23 and co-workers recently discovered that thymine starvation initially leads to the accumulation 24 of ssDNA gaps, which are subsequently converted to DSBs in an ROS-dependent process5 25 (Figure 1b). In cells treated with ROS scavengers, gaps are not converted to DSBs and 26 cells are tolerant to thymine starvation5. 27 Several lines of evidence implicate pol IV in ROS-dependent DSB formation and/or 28 processing. Pol IV efficiently incorporates 8-oxo-dGTP into DNA in vitro7. Over-expression of 29 pol IV is lethal to cells and the lethality is ROS-dependent4,7,19. Cells lacking pol IV are 30 partially protected against killing by ampicillin under conditions where the nucleotide pool 31 becomes oxidised7. These observations are suggestive of pol IV playing a role in the BER- 32 dependent mechanism for DSB formation4 (Figure 1a). Other studies indicate that pol IV has 33 a role in the repair of DSBs6,9,10,12,13,20–26. First, pol IV physically interacts with the RecA 34 recombinase; a key player in DSB repair (DSBR)24. Second, when expressed from a low- 35 copy plasmid, fluorescently labelled pol IV colocalises with RecA extensively in cells treated 36 with DNA-damaging agents10. Pol IV colocalises at sites of induced DSBs in cells10. Third, 37 genetic studies reveal that the gene encoding pol IV, dinB, is required for both induced and 38 spontaneous error-prone DSBR6,9,12,13,22,23. Fourth, intermediates of DSBR known as 39 recombination D-loops are efficiently utilised as substrates by pol IV in vitro25. Interestingly, 40 pol IV’s mutagenic potential is modulated by interaction with the recombinase RecA20,21,24,26. 41 Notably, the cellular concentration of pol IV increases significantly upon induction of the SOS 42 response27,28. Single-stranded DNA formed during DSBR (through RecBCD-dependent end- 43 resection) is a major trigger for SOS induction under some conditions29–31. 44 Here, we used single-molecule fluorescence microscopy to investigate whether ROS, 45 and the DSBs they create, influence pol IV activity in cells. We made use of two antibiotics 46 for which killing is known to involve ROS generation; ciprofloxacin and trimethoprim (Figure 47 1a)32–35. For ciprofloxacin, a DNA gyrase inhibitor, a second, ROS-independent pathway 48 exists in which gyrase-stabilised cleavage complexes dissociate, creating a DSB (Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/533422; this version posted January 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 1b)32,33,36,37. During antibiotic treatment, we would expect that some pol IV binding would be 2 dependent on ROS and/or DSB processing while others would be independent of these 3 factors. Throughout the study, we used dimethyl sulfoxide (DMSO) to create low ROS 4 conditions. DMSO is an effective scavenger of antibiotic-induced ROS5,38. In agreement with 5 the scheme in Figure 1a, co-treatment with DMSO increases survival in cells exposed to 6 both ciprofloxacin and trimethoprim5,38 (Supplementary figure 1,2). 7 To compare pol IV behaviour under high-ROS (no DMSO) and low-ROS (DMSO 8 added) conditions, we recorded time-lapse movies (180 min duration)28,39 of cells treated 9 with i) ciprofloxacin alone, ii) ciprofloxacin and DMSO in combination, iii) trimethoprim alone 10 or iv) trimethoprim and DMSO in combination (Figure 2). Following antibiotic addition, we 11 recorded time-lapse movies capturing fluorescence from a functional DinB-YPet fusion28. 12 Cells treated with ciprofloxacin became filamentous and showed a clear increase in DinB- 13 YPet intensity (Figure 2a,b; Supplementary figure 3). This is consistent with our previous 14 study in which we measured a six-fold increase in intracellular DinB-YPet (pol IV) 15 concentrations from 6 ± 1 nM prior to treatment to 34 ± 3 nM 180 min after ciprofloxacin 16 addition28. Interestingly, inclusion of the ROS scavenger DMSO led to a significant reduction 17 in ciprofloxacin-induced up-regulation of pol IV levels; 180 min after ciprofloxacin addition, 18 cellular DinB-YPet intensities were increased above basal levels by less than 3 fold (Figure 19 2a,b), corresponding to a final concentration of ~17 nM (see Materials and Methods). 20 Treatment with trimethoprim alone led to a significant increase in DinB-YPet fluorescence; 21 180 min after trimethoprim addition the mean fluorescence intensity increased by 50% of 22 ciprofloxacin-alone levels (Figure 2a,b), corresponding to a final intracellular pol IV 23 concentration of ~20 nM. Inclusion of DMSO led to a significant reduction in trimethoprim- 24 induced up-regulation; cell-associated DinB-YPet intensities increased by only 11% 25 compared with ciprofloxacin-alone treatment, corresponding to a final pol IV concentration of 26 ~9 nM. Thus, for both the ciprofloxacin and trimethoprim treatments, addition of the ROS 27 scavenger DMSO significantly reduces the up-regulation of pol IV in response to antibiotic 28 treatment. 29 Ciprofloxacin-treated cells also produced multiple DinB-YPet foci (Figure 2c). These 30 foci require the catalytic activity of pol IV and are produced as individual DinB-YPet 31 molecules bind to DNA and thus experience decreased diffusional mobility28, a phenomenon 32 known as detection by localisation40. Prior to the addition of ciprofloxacin, cells contained on 33 average 0.6 ± 0.2 foci per cell in the absence of DMSO, and 0.4 ± 0.1 foci per cell in the 34 presence of DMSO (Figure 2c). Following treatment with ciprofloxacin alone, the number of 35 foci steadily increased. By 180 min, cells had 4.2 ± 1.1 foci per cell. Upon ciprofloxacin- 36 DMSO treatment, cells contained 1.8 ± 0.4 foci per cell; a significant reduction compared to 37 ciprofloxacin-alone measurements. Addition of DMSO reduced the number of DinB-YPet foci 38 by >50%. Prior to the addition of trimethoprim, cells contained on average 0.5 ± 0.1 foci in 39 the absence of DMSO and 0.4 ± 0.1 foci in the presence of DMSO. Trimethoprim-alone 40 treatment induced a slight increase in the number of DinB-YPet foci with 0.9 ± 0.2 per cell at 41 180 min. This is lower than the number of foci observed for ciprofloxacin-DMSO treatment 42 (1.8 ± 0.4 per cell), despite the measured pol IV concentration being marginally higher after 43 trimethoprim-alone treatment (Figure 2b).
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