Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

Shee, Chandan; Cox, Ben D.; Gu, Franklin; Luengas, Elizabeth M.; Joshi, Mohan C.; Chiu, Li Ya; Magnan, David; Halliday, Jennifer A.; Frisch, Ryan L.; Gibson, James W.; Nehring, Ralf B.; Huong, G.; Hernandez, M. Rosario; Li, Lei; Herman, Christophe; Hastings, P.J.; Bates, David; Harris, Reuben S.; Miller, Kyle M.; Rosenberg, Susan M.

doi:10.7554/elife.01222

Cited by 117 publications

(194 citation statements)

References 86 publications

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“…Motivated by our nucleotide results and to test further the hypothesis that bactericidal antibiotics induce DSBs in the bacterial chromosome, we took advantage of a novel engineered fluorescent protein-based probe (Shee et al, 2013). This method relies on a fusion of GFP to the Gam protein from phage Mu that robustly binds to DSBs when expressed in E. coli .…”

Section: Resultsmentioning

confidence: 99%

“…To visually profile the formation of DSB foci, we treated cells expressing Gam-GFP with H 2 O 2 , which is known to induce DSBs (Friedberg et al, 1996), or with Amp, Kan, or Nor. Expression of Gam-GFP allows reliable detection and enumeration of DSBs, which appear as fluorescent GFP foci (Shee et al, 2013). As some antibiotics cause protein misfolding and polar aggregation, cells were stained with DAPI prior to imaging, and only foci co-localized with DAPI in the deconvolved three-dimensional image were quantified as DSB foci.…”

Section: Resultsmentioning

confidence: 99%

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Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

et al. 2015

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Summary Understanding how antibiotics impact bacterial metabolism may provide insight into their mechanisms of action and could lead to enhanced therapeutic methodologies. Here, we profiled the metabolome of Escherichia coli after treatment with three different classes of bactericidal antibiotics (beta-lactams, aminoglycosides, quinolones). These treatments induced a similar set of metabolic changes after 30 minutes that then diverged into more distinct profiles at later timepoints. The most striking changes corresponded to elevated concentrations of central carbon metabolites, active breakdown of the nucleotide pool, reduced lipid levels, and evidence of an elevated redox state. We examined potential end-target consequences of these metabolic perturbations and found that antibiotic-treated cells exhibited cytotoxic changes indicative of oxidative stress, including higher levels of protein carbonylation, malondialdehyde adducts, nucleotide oxidation, and double-strand DNA breaks. This work shows that bactericidal antibiotics induce a complex set of metabolic changes that are correlated with the buildup of toxic metabolic by-products.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

et al. 2015

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“…During normal cell growth, DSBs are common and, if unrepaired, are lethal. Estimates of the number of spontaneous DSBs per generation in a growing E. coli cell range from 0.01 to 0.3 (75,76). As mentioned above, most DSBs are repaired by the RecA-RecBCD homologous-recombination pathway, followed by recruitment of primasome proteins to restart replication (76).…”

Section: Discussionmentioning

confidence: 99%

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

et al. 2015

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Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNAdamaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ϳ60% of the foci consisted of three Pol IV molecules, while ϳ40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ϳ70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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“…Laser microirradiation was carried out with a FluoView 1000 confocal microscope (Olympus) as described (Shee et al 2013). Briefly, cells were grown on glass-bottomed dishes (Willco Wells) and presensitized with 10 mM 5-bromo-29-deoxyuridine (BrdU) for 24 h at 37°C.…”

Section: Laser Microirradiation and Microscopy Analysismentioning

confidence: 99%

Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination

et al. 2015

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How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary ''readers'' of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.

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Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

Cited by 117 publications

References 86 publications

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination

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