Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions

AbstractDNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in “clustered” oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in “real time” by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

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Antibiotic amoxicillin induces DNA lesions in mammalian cells possibly via the reactive oxygen species

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2007.02.005 ◽

2007 ◽

Vol 629 (2) ◽

pp. 133-139 ◽

Cited By ~ 19

Author(s):

Pei-Yi Li ◽

Yu-Ching Chang ◽

Bor-Show Tzang ◽

Chung-Chu Chen ◽

Yin-Chang Liu

Keyword(s):

Reactive Oxygen Species ◽

Mammalian Cells ◽

Reactive Oxygen ◽

Dna Lesions ◽

Oxygen Species

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Excessive Reactive Oxygen Species and Exotic DNA Lesions as an Exploitable Liability

Biochemistry ◽

10.1021/acs.biochem.6b00703 ◽

2016 ◽

Vol 55 (38) ◽

pp. 5341-5352 ◽

Cited By ~ 27

Author(s):

Safnas F. AbdulSalam ◽

Fathima Shazna Thowfeik ◽

Edward J. Merino

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Dna Lesions ◽

Oxygen Species

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Reactive oxygen species play a dominant role in all pathways of rapid quinolone-mediated killing

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz485 ◽

2019 ◽

Vol 75 (3) ◽

pp. 576-585 ◽

Cited By ~ 6

Author(s):

Yuzhi Hong ◽

Qiming Li ◽

Qiong Gao ◽

Jianping Xie ◽

Haihui Huang ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Dominant Role ◽

Reactive Oxygen ◽

Dna Lesions ◽

Dominant Factor ◽

Oxygen Species ◽

Protective Effects ◽

Dna Breaks ◽

E Coli ◽

Ros Accumulation

Abstract Background Quinolones have been thought to rapidly kill bacteria in two ways: (i) quinolone-topoisomerase-DNA lesions stimulate the accumulation of toxic reactive oxygen species (ROS); and (ii) the lesions directly cause lethal DNA breaks. Traditional killing assays may have underestimated the ROS contribution by overlooking the possibility that ROS continue to accumulate and kill cells on drug-free agar after quinolone removal. Methods Quinolone-induced, ROS-mediated killing of Escherichia coli was measured by plating post-treatment samples on agar with/without anti-ROS agents. Results When E. coli cultures were treated with ciprofloxacin or moxifloxacin in the presence of chloramphenicol (to accentuate DNA-break-mediated killing), lethal activity, revealed by plating on quinolone-free agar, was inhibited by supplementing agar with ROS-mitigating agents. Moreover, norfloxacin-mediated lethality, observed with cells suspended in saline, was blocked by inhibitors of ROS accumulation and exacerbated by a katG catalase deficiency that impairs peroxide detoxification. Unlike WT cells, the katG mutant was killed by nalidixic acid or norfloxacin with chloramphenicol present and by nalidixic or oxolinic acid with cells suspended in saline. ROS accumulated after quinolone removal with cultures either co-treated with chloramphenicol or suspended in saline. Deficiencies in recA or recB reduced the protective effects of ROS-mitigating agents, supporting the idea that repair of quinolone-mediated DNA lesions suppresses the direct lethal effects of such lesions. Conclusions ROS are the dominant factor in all modes of quinolone-mediated lethality, as quinolone-mediated primary DNA lesions are insufficient to kill without triggering ROS accumulation. ROS-stimulating adjuvants may enhance the lethality of quinolones and perhaps other antimicrobials.

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Bulky DNA Lesions Induced by Reactive Oxygen Species

Chemical Research in Toxicology ◽

10.1021/tx700411g ◽

2008 ◽

Vol 21 (2) ◽

pp. 276-281 ◽

Cited By ~ 103

Author(s):

Yinsheng Wang

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Dna Lesions ◽

Oxygen Species ◽

Bulky Dna Lesions

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Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage

PLoS ONE ◽

10.1371/journal.pone.0237759 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0237759

Author(s):

Nealia C. M. House ◽

Ramya Parasuram ◽

Jacob V. Layer ◽

Brendan D. Price

Keyword(s):

Reactive Oxygen Species ◽

Dna Damage ◽

Oxidative Dna Damage ◽

Light Exposure ◽

Green Light ◽

Reactive Oxygen ◽

Dna Lesions ◽

Oxygen Species ◽

Dna Breaks ◽

Site Specific

DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in “clustered” oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in “real time” by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

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Mitochondria as a source of reactive oxygen species

Biochemical Journal ◽

10.1042/bj20090056 ◽

2009 ◽

pp. c3 ◽

Cited By ~ 1

Author(s):

Helena M. Cochemé ◽

Michael P. Murphy

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Oxygen Species

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Clinical aspects of reactive oxygen and nitrogen species

Biochemical Society Symposium ◽

10.1042/bss0710121 ◽

2004 ◽

Vol 71 ◽

pp. 121-133 ◽

Cited By ~ 25

Author(s):

Ascan Warnholtz ◽

Maria Wendt ◽

Michael August ◽

Thomas Münzel

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Risk Factor ◽

Endothelial Dysfunction ◽

Vascular Tissue ◽

Rapid Development ◽

Reactive Oxygen ◽

Clinical Aspects ◽

No Production ◽

Oxygen Species

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.

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