Hydrogen peroxide -dependent and -independent DNA damage and repair

DNA damage and repair at environmental concentrations of hydrogen peroxide E. coli responds to hydrogen peroxide (H2O2) by inducing defenses that protect H2O2-sensitive enzymes. DNA is believed to be another important target of oxidation, and E. coli contains enzymes that can repair oxidative lesions in vitro. However, those enzymes are not known to be induced by H2O2, and experiments have indicated that they are not necessary for the cell to withstand natural (low-micromolar) H2O2 concentrations. In this study we used H2O2-scavenging mutants to impose controlled doses of H2O2 for extended time. Transcriptomic analysis revealed that in the presence of 1 µM cytoplasmic H2O2, the OxyR transcription factor induced xthA, encoding exonuclease III. The xthA mutants survived a conventional 15-minute exposure to even 100 times this level of H2O2. However, when these mutants were exposed to 1 µM H2O2 for hours, they accumulated DNA lesions, failed to propagate, and eventually died. Although endonuclease III (nth) was not induced, nth mutants struggled to grow. Low-grade H2O2 stress also activated the SOS regulon, and when this induction was blocked, cell replication stopped. Collectively, these data indicate that physiological levels of H2O2 are a real threat to DNA, and the engagement of the base-excision-repair and SOS systems is necessary to enable propagation during protracted stress. Does the lethal action of streptonigrin involve production of diffusible superoxide/hydrogen peroxide? Streptonigrin (SN) is an aminoquinone antibiotic produced by Streptomyces flocculus. This drug is known to cause DNA damage, and it requires both iron and oxygen for its toxicity. Hence it has been proposed that the molecular mechanism for the toxicity of SN is through redox cycling of electrons to oxygen, resulting in the production of reactive oxygen species (ROS). However, we suspect that if ROS were being generated by SN, the cellular oxidative stress response systems could reduce the efficacy of SN. Yet, SN-treated cells die rapidly. Thus, we hypothesize that SN toxicity is not dependent on the formation of ROS. Using E. coli as a model organism, we have verified the dependence of SN on oxygen and iron for causing DNA damage. However, unlike other quinone drugs (for example, menadione) which are known to elevate intracellular levels of ROS, we do not find any evidence of ROS formation in SN-treated cells. There is no increase in oxygen consumption of SN-treated cells, no elevated production of hydrogen peroxide, no induction of ROS defense systems, and no increase in sensitivity of ROS scavenging mutants to SN. Using an in vitro DNA damaging system, we also confirmed that SN-mediated DNA strand break occurs even in the absence of ROS like hydrogen peroxide, establishing that SN toxicity is not dependent on increase in H2O2 levels. We suspect that the DNA damage caused by SN could occur through the formation of a high-valence oxidant like a ferryl radical species. Thus, by avoiding the increase in ROS production inside a cell, SN evades cellular defenses to cause damage on the DNA.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Gupta, Anshika

Contributors dc:contributor

• Imlay, James
• Kuzminov, Andrei
• Vanderpool, Carin
• Olsen, Gary

• Oxidative Stress
• Dna Damage
• Streptonigrin
• Dna Repair
• Hydrogen Peroxide
• Exonuclease Iii

Statement dc:rights

• Copyright 2023 Anshika Gupta

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/120317

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F120317